Nucleic acid preparation compositions and methods

ABSTRACT

Provided herein are methods and compositions to extract and enrich by, physical separation or amplification, relatively short nucleic acids from a nucleic acid composition containing a high background of longer nucleic acids (e.g., host or maternal nucleic acids; genomic nucleic acid and the like).

RELATED PATENT APPLICATION(S)

This application is a continuation application of U.S. patentapplication Ser. No. 15/409,189, filed on Jan. 18, 2017, entitledNUCLEIC ACID PREPARATION COMPOSITIONS AND METHODS, naming MicheleElizabeth Wsniewski, William Hang Kwong, Firouz Mohsenian, and Jian-HuaDing as applicants and inventors, and designated by attorney docket no.PLA-6026-CT2, which is a continuation application of U.S. patentapplication Ser. No. 14/296,732, filed on Jun. 5, 2014, now U.S. Pat.No. 9,580,741, entitled NUCLEIC ACID PREPARATION COMPOSITIONS ANDMETHODS, naming Michele Elizabeth Wisniewski, William Hang Kwong, FirouzMohsenian, and Jian-Hua Ding as applicants and inventors, and designatedby attorney docket no. PLA-6026-CT, which is a continuation applicationof U.S. patent application Ser. No. 13/262,624, now U.S. Pat. No.8,771,948, filed on Mar. 1, 2012, entitled NUCLEIC ACID PREPARATIONCOMPOSITIONS AND METHODS, naming Michele Elizabeth Wisniewski, WilliamHang Kwong, Firouz Mohsenian, and Jian-Hua Ding as applicants andinventors, and designated by attorney docket no. PLA-6026-US, which is anational stage of international patent application numberPCT/US2010/029653 filed on Apr. 1, 2010, entitled NUCLEIC ACIDPREPARATION COMPOSITIONS AND METHODS, naming Michele ElizabethWisniewski, William Hang Kwong, Firouz Mohsenian, and Jian-Hua Ding asapplicants and inventors, and designated by attorney docket no.PLA-6026-PC, which claims the benefit of U.S. provisional patentapplication No. 61/166,671 filed on Apr. 3, 2009, entitled NUCLEIC ACIDPREPARATION COMPOSITIONS AND METHODS, naming Michele ElizabethWisniewski, William Hang Kwong, Firouz Mohsenian, and Jian-Hua Ding asinventors and designated by attorney docket no. PLA-6026-PV. The entirecontents of the foregoing patent applications are incorporated herein byreference, including, without limitation, all text, tables and drawings.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 28, 2014, isnamed SEQ-6026-CT_SL.txt and is 9,028 bytes in size.

FIELD OF THE TECHNOLOGY

The technology relates in part to compositions and methods for nucleicacid preparation and enrichment.

BACKGROUND

The isolation and subsequent amplification of nucleic acids play acentral role in molecular biology. Isolated, purified nucleic acids maybe used, inter alia, as a starting material for diagnosis and prognosisof diseases or disorders. Therefore, the isolation of nucleic acids,particularly by non-invasive means, is of particular importance for usein genetic analyses.

Current methods for the extraction of nucleic acids include the use oforganic-based methods (e.g., phenol/chloroform/isoamyl alcohol), orcapitalize upon ion interaction of nucleic acids in an aqueous solution(e.g., salting out in combination with alcohol, solution pH andtemperature) alone or in combination with anion exchange chromatographyor cation exchange chromatography. Organic-based methods employ the useof phenol/chloroform/isoamyl alcohol or variations thereof for isolatingDNA, but have serious disadvantages, namely the processes are verytime-consuming, require considerable experimental effort, and areassociated with an acute risk of exposure to toxic substances to thosecarrying out the isolation. Chromatography-based methods increaseflexibility and automation since these methods can be used incombination with multiple matrices (e.g., membranes, latex, magneticbeads, micro-titer plate, etc.) and in the presence or absence ofligands (e.g., DEAE, silica, acrylamide, etc.). However, these methodsare better suited to extract larger strands of nucleic acids to ensuregreater success in downstream analysis.

Previously, the recovery of smaller, fragmented nucleic acids frombiological samples was considered unimportant, and extraction methodswere designed to isolate large, undegraded nucleic acid molecules.Recently, however, it is shorter base pair nucleic acids (e.g., highlydegraded RNA or mRNA and apoptotic DNA) that have been shown to behighly informative for a wide range of applications, including prenataldiagnostics and the study of apoptotic DNA from host or non-hostsources.

SUMMARY

The present technology provides improved nucleic acid preparationcompositions and methods suitable for enrichment, isolation and analysisof relatively short nucleic acid species targets, sometimes found incell free or substantially cell free biological compositions containingmixed compositions (e.g., viral nucleic acid in host background, fetalnucleic acid in maternal background, mixed nucleic acid populations fromenvironmental samples, and the like), and often associated with variousdisease conditions or apoptotic cellular events (e.g., cancers and cellproliferative disorders, prenatal or neonatal diseases, geneticabnormalities, and programmed cell death events). The relatively shortnucleic acid species targets, which can represent degraded orfractionated nucleic acids, can also be used for haplotyping andgenotyping analysis, such as fetal genotyping for example.

Methods and compositions described herein are useful for size selectionof nucleic acids, in a simple, cost effective manner that also can becompatible with automated and high throughput processes and apparatus.Methods and compositions provided herein are useful for enriching orextracting a target nucleic acid from a cell free or substantially cellfree biological composition containing a mixture of non-target nucleicacids, based on the size of the nucleic acid, where the target nucleicacid is of a different size, and often is smaller, than the non-targetnucleic acid.

Thus provided in some embodiments is a method for enriching relativelyshort nucleic acid from a nucleic acid composition, which comprises, (a)contacting nucleic acid of a nucleic acid composition with a solid phaseunder association conditions, wherein: (i) the nucleic acid of thenucleic acid composition comprises relatively short nucleic acid andrelatively long nucleic acid, (ii) the relatively short nucleic acid isabout 300 base pairs or less, and (iii) the relatively long nucleic acidis larger than about 300 base pairs; whereby the relatively shortnucleic acid and the relatively long nucleic acid are associated withthe solid phase; (b) introducing the solid phase after (a) todissociation conditions that comprise a volume exclusion agent and asalt, wherein: (i) the salt is not a chaotropic salt, and (ii) therelatively short nucleic acid preferentially dissociates from the solidphase under the dissociation conditions as compared to the relativelylong nucleic, thereby yielding dissociated nucleic acid; and (c)separating the dissociated nucleic acid from the solid phase, wherebythe relatively short nucleic acid is enriched in the dissociated nucleicacid relative to the relatively long nucleic acid in the nucleic acidcomposition. In some embodiments, the dissociated nucleic acid comprisesribonucleic acid (RNA), and in certain embodiments consists essentiallyof RNA. In some embodiments, the dissociated nucleic acid comprisesdeoxyribonucleic acid (DNA), and in certain embodiments consistsessentially of DNA.

In some embodiments, about 30% to about 90% of the nucleic acid of thenucleic acid composition associates with the solid phase. In certainembodiments, about 60% of the nucleic acid of the nucleic acidcomposition associates with the solid phase. In some embodiments, themethod further comprises washing the solid phase after (a). In certainembodiments, the solid phase is washed under conditions that removematerial of the nucleic acid composition not associated with the solidphase from the solid phase. In some embodiments, the solid phase iswashed under conditions that dissociate any non-nucleic acid material ofthe nucleic acid composition from the solid phase. In certainembodiments, the wash comprises an alcohol solution.

In some embodiments, the association conditions comprise a C1-C6 alkylalcohol, and in certain embodiments the association conditions consistessentially of a C1-C6 alkyl alcohol. In certain embodiments, theassociation conditions do not comprise a C1-C6 alkyl alcohol. In someembodiments, the alcohol comprises ethanol. In some embodiments, theassociation conditions comprise a salt. In certain embodiments, the saltcomprises a chaotropic salt, an ionic salt or combination thereof. Insome embodiments using ionic salts or a combination of salts, the ionicsalt is sodium chloride. In certain embodiments, the associationconditions consist essentially of a salt. In some embodiments, theassociation conditions do not comprise a salt. In some embodiments, theassociation conditions do not comprise a chaotropic agent (e.g., nochaotropic salt).

In certain embodiments, the association conditions comprise a volumeexclusion agent. In some embodiments, the association conditions consistessentially of a volume exclusion agent. In certain embodiments, thevolume exclusion agent comprises a polyalkyl alcohol (e.g., polyalkylglycol or polyethylene glycol), dextran, Ficoll, polyvinyl pyrollidoneor combination thereof. In some embodiments, the polyalkyl alcohol ispolyethylene glycol (PEG), and in certain embodiments the PEG is PEG8000. In some embodiments, the association conditions do not comprise avolume exclusion agent. In some embodiments, the association conditionsdo not comprise polyethylene glycol.

In some embodiments, the dissociation conditions comprise about 0.25M toabout 0.5M of the ionic salt. In certain embodiments, the dissociationconditions comprise about 10% PEG. In some embodiments, the salt and thevolume exclusion agent are present in the dissociation conditions atconcentrations according to Table 1 (presented below in Example 3). Insome embodiments, the dissociation conditions do not comprise achaotropic agent (e.g., no chaotropic salt). In certain embodiments, therelatively short nucleic acid preferentially dissociates from the solidphase under the association conditions as compared to the relativelylong nucleic acid at a ratio of about 1.05 to about 5 relatively shortnucleic acid to relatively long nucleic acid. In certain embodiments,the relatively short nucleic acid is enriched about 10% to about 45% inthe dissociated nucleic acid relative to in the nucleic acidcomposition.

In some embodiments, the solid phase is paramagnetic and the dissociatednucleic acid is separated from the solid phase by a magnet or magneticfield. In certain embodiments, the solid phase is separated from thedissociated nucleic acid by centrifugation. In certain embodiments, thesolid phase is not paramagnetic. In some embodiments, the solid phase isseparated from the dissociated nucleic acid by transferring thedissociated nucleic acid to an environment that does not contain thesolid phase used in (a) of the method described above. In certainembodiments, the solid phase is separated from the dissociated nucleicacid by transferring the solid phase to an environment that does notcontain the dissociated nucleic acid. In certain embodiments, theenvironment is a vessel. The term “vessel” as used herein, refers to anycontainer, plate (e.g., multiwell plate), tube, and the like, suitablefor carrying out the methods described herein.

In some embodiments, the method further comprises associating thedissociated nucleic acid to a second solid phase. In certainembodiments, the method further comprises dissociating the dissociatednucleic acid from the second solid phase, thereby releasing thedissociated nucleic acid from the second solid phase. In someembodiments, the method further comprises analyzing the dissociatednucleic acid and/or nucleic acid associated with the solid phase after(c) by mass spectrometry. In certain embodiments, the method furthercomprises contacting the dissociated nucleic acid and/or nucleic acidassociated with the solid phase after (c) with an oligonucleotide thathybridizes to the dissociated nucleic acid and is extended underextension conditions, thereby yielding extended oligonucleotide.

In some embodiments, the method further comprises amplifying thedissociated nucleic acid and/or the nucleic acid associated with thesolid phase after (c), thereby yielding amplified product. In certainembodiments, the method further comprises contacting the amplifiedproduct with an oligonucleotide that hybridizes to the amplified productand is extended under extension conditions, thereby yielding extendedoligonucleotide. In some embodiments, the method further comprisesanalyzing the extended oligonucleotide or the amplified product. Incertain embodiments, the extended oligonucleotide or the amplifiedproduct is analyzed by mass spectrometry.

In some embodiments, the nucleic acid composition is a biologicalcomposition. In certain embodiments, the biological composition is asubstantially cell-free biological composition. In certain embodiments,the nucleic acid is cell free nucleic acid. In some embodiments, thesubstantially cell free biological composition is from a pregnantfemale. In certain embodiments, the pregnant female is in the firsttrimester of pregnancy. In some embodiments, the substantially cell-freebiological composition is blood plasma and in certain embodiments thesubstantially cell-free biological composition is blood serum. Incertain embodiments, the substantially cell-free biological compositionis urine.

In some embodiments, the method further comprises detecting the presenceor absence of fetal nucleic acid, and in some embodiments comprisesdetecting the presence or absence of a fetal-specific nucleotidesequence. In certain embodiments, the fetal-specific nucleotide sequenceis a Y-chromosome sequence. In some embodiments, the fetal-specificnucleotide sequence is a mRNA sequence. In some embodiments, thefetal-specific nucleotide sequence is labeled. In certain embodiments,the method further comprises quantifying the labeled fetal-specificnucleotide sequence. In certain embodiments, the method furthercomprises detecting the presence or absence of a prenatal disorder. Insome embodiments, the prenatal disorder is a chromosome abnormality. Incertain embodiments, the chromosome abnormality is a trisomy. In someembodiments, the trisomy is trisomy 21, trisomy 18, trisomy 13 orcombination thereof. In certain embodiments, the method furthercomprises detecting the presence or absence of a cell proliferationdisorder. In some embodiments, the cell proliferation disorder is acancer.

Certain embodiments are described further in the following description,examples, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings illustrate embodiments of the technology and are notlimiting. For clarity and ease of illustration, the drawings are notmade to scale and, in some instances, various aspects may be shownexaggerated or enlarged to facilitate an understanding of particularembodiments.

FIG. 1 illustrates the results of gel electrophoresis of nucleic acids,in a 1-kilobase size ladder, extracted or enriched after association andsize selective dissociation of nucleic acids. FIG. 1A show the resultsof sample nucleic acid association to solid support followed bydissociation to illustrate the size distribution of fragments in thesample. FIG. 1B show the results of elution of nucleic acids from thesolid support after an initial size selection was performed and theinitially eluted fragments were separated from the material still boundto the solid support. The initial size selection dissociates the smallerfragments (shown in FIG. 10), leaving behind larger fragments, accordingto the salt concentration used. FIG. 10 illustrates the sizedistribution of the nucleic acids initially dissociated, according tothe dissociation conditions given above each gel lane.

FIG. 2 shows the percent male fetus DNA relative to total DNA isolatefrom the serum of a pregnant female, and the recovery of small fragmentsat various salt concentrations as compared to the recovery of largefragments at the same salt concentrations. The percent fold enrichment(e.g., approximately 30%) can be calculated from the data presented inFIG. 2, as described in Example 2. Enrichment was performed using threedifferent salt titrations 0.375M NaCl/10% PEG, 0.5M NaCl/10% PEG, and 1MNaCl/10% PEG, which selects for less than 500 base pairs, less than 400base pairs, and less than 300 base pairs, respectively. In FIG. 2,“short” fragment refers to DNA fragments less than the designated cutoffsize provided, for example, less than 500 base pairs at 0.375M NaCl/10%PEG, less than 400 base pairs at 0.5M NaCl/10% PEG, and less than 300base pairs at 1M NaCl/10% PEG.

DETAILED DESCRIPTION

The presence of cell-free nucleic acid in peripheral blood is a wellestablished phenomenon. While cell-free nucleic acid may originate fromseveral sources, it has been demonstrated that one source of circulatingextracellular nucleic acid originates from programmed cell death, alsoknown as apoptosis. The source of nucleic acid that arise as a result ofapoptosis may be found in many body fluids and originate from severalsources, including, but not limited to, normal programmed cell death inthe host, induced programmed cell death in the case of an autoimmunedisease, septic shock, neoplasms (malignant or non-malignant), ornon-host sources such as an allograft (transplanted tissue), or thefetus or placenta of a pregnant woman. The applications for thedetection, extraction and relative enrichment of extracellular nucleicacid from peripheral blood or other body fluids are widespread and mayinclude inter alia, non-invasive prenatal diagnosis, cancer diagnostics,pathogen detection, auto-immune response and allograft rejection.

In some embodiments, methods and compositions are provided that enableenrichment and/or extraction of relatively short target nucleic acidfragments, of specific size ranges (e.g., 50-500 nucleotides or basepairs, and more specifically 50 to 200 nucleotides or base pairs, forexample, and herein referred to as “target” or “sample” nucleic acid),contained within a nucleic acid composition of mixed fragment sizes(e.g., 1 to 100,000 nucleotides or base pairs (bp), or more).

The enrichment and/or extraction of the target nucleic acid can beaccomplished by a partial, or complete, physical separation of thetarget nucleic acid from the rest of the nucleic acid in the nucleicacid composition. More specifically, the methods and compositionsdescribed herein, are useful for the selective extraction and relativeenrichment, based on size discrimination, of nucleic acids of in therange of about 50 to about 500 nucleotides or base pairs, and morespecifically about 50 to about 200 nucleotides or base pairs, in a highbackground of genomic nucleic acids (herein referred to as “non-target”nucleic acid). The methods and compositions described herein lead to arelatively enriched fraction of nucleic acids that has a higherconcentration of smaller nucleic acids, where the smaller nucleic acidssometimes contain target nucleic acids. In some embodiments, furtherenrichment of the specific target nucleic acids can be accomplished byamplification of the specific size selected target nucleic acidsequences using amplification procedures known in the art or describedbelow.

Disorders

Nucleic acid prepared using methods and compositions described hereincan be utilized to detect the presence or absence of one or moreprenatal or neonatal disorders. Non-limiting examples of prenatal andneonatal disorders include achondroplasia, Angelman syndrome, Cockaynesyndrome, cystic fibrosis (autosomal recessive), congenital adrenalhyperplasia (autosomal recessive), DiGeorge syndrome, Duchenne'smuscular dystrophy, (X-linked recessive), hemophilia A (X-linkedrecessive), alpha- and beta-thalassemia (autosomal recessive), fragile Xsyndrome (X-linked dominant), polycystic kidney disease (adult type;autosomal dominant), sickle cell anemia (autosomal recessive), Marfansyndrome, Prader-Wlli syndrome, Waardenburg syndrome, Tay-Sachs disease(autosomal) and the like.

A prenatal or neonatal disorder in some embodiments is a chromosomeabnormality. In certain embodiments chromosome abnormalities include,without limitation, a gain or loss of an entire chromosome or a regionof a chromosome comprising one or more genes. Chromosome abnormalitiesinclude monosomies, trisomies, polysomies, loss of heterozygosity,deletions and/or duplications of one or more nucleotide sequences (e.g.,one or more genes), including deletions and duplications caused byunbalanced translocations in some embodiments. The terms “aneuploidy”and “aneuploid” as used herein refer to an abnormal number ofchromosomes in cells of an organism. As different organisms have widelyvarying chromosome complements, the term “aneuploidy” does not refer toa particular number of chromosomes, but rather to the situation in whichthe chromosome content within a given cell or cells of an organism isabnormal.

The term “monosomy” as used herein refers to lack of one chromosome ofthe normal complement. Partial monosomy can occur in unbalancedtranslocations or deletions, in which only a portion of the chromosomeis present in a single copy (see deletion (genetics)). Monosomy of sexchromosomes (45, X) causes Turner syndrome.

The term “disomy” refers to the presence of two copies of a chromosome.For organisms such as humans that have two copies of each chromosome(those that are diploid or “euploid”), it is the normal condition. Fororganisms that normally have three or more copies of each chromosome(those that are triploid or above), disomy is an aneuploid chromosomecomplement. In uniparental disomy, both copies of a chromosome come fromthe same parent (with no contribution from the other parent).

The term “trisomy” refers to the presence of three copies, instead ofthe normal two, of a particular chromosome. The presence of an extrachromosome 21, which is found in Down syndrome, is called trisomy 21.Trisomy 18 and Trisomy 13 are the two other autosomal trisomiesrecognized in live-born humans. Trisomy of sex chromosomes can be seenin females (47, XXX) or males (47, XXY which is found in Klinefelter'ssyndrome; or 47,XYY).

The terms “tetrasomy” and “pentasomy” as used herein refer to thepresence of four or five copies of a chromosome, respectively. Althoughrarely seen with autosomes, sex chromosome tetrasomy and pentasomy havebeen reported in humans, including)(XXX, XXXY, XXYY, XYYY, XXXXX, XXXXY,XXXYY, XXYYY and XYYYY.

Chromosome abnormalities can be caused by a variety of mechanisms.Mechanisms include, but are not limited to (i) nondisjunction occurringas the result of a weakened mitotic checkpoint, (ii) inactive mitoticcheckpoints causing non-disjunction at multiple chromosomes, (iii)merotelic attachment occurring when one kinetochore is attached to bothmitotic spindle poles, (iv) a multipolar spindle forming when more thantwo spindle poles form, (v) a monopolar spindle forming when only asingle spindle pole forms, and (vi) a tetraploid intermediate occurringas an end result of the monopolar spindle mechanism.

The terms “partial monosomy” and “partial trisomy” as used herein referto an imbalance of genetic material caused by loss or gain of part of achromosome. A partial monosomy or partial trisomy can result from anunbalanced translocation, where an individual carries a derivativechromosome formed through the breakage and fusion of two differentchromosomes. In this situation, the individual would have three copiesof part of one chromosome (two normal copies and the portion that existson the derivative chromosome) and only one copy of part of the otherchromosome involved in the derivative chromosome.

The term “mosaicism” as used herein refers to aneuploidy in some cells,but not all cells, of an organism. Certain chromosome abnormalities canexist as mosaic and non-mosaic chromosome abnormalities. For example,certain trisomy 21 individals have mosaic Down syndrome and some havenon-mosaic Down syndrome. Different mechanisms can lead to mosaicism.For example, (i) an initial zygote may have three 21st chromosomes,which normally would result in simple trisomy 21, but during the courseof cell division one or more cell lines lost one of the 21stchromosomes; and (ii) an initial zygote may have two 21st chromosomes,but during the course of cell division one of the 21st chromosomes wereduplicated. Somatic mosaicism most likely occurs through mechanismsdistinct from those typically associated with genetic syndromesinvolving complete or mosaic aneuploidy. Somatic mosaicism has beenidentified in certain types of cancers and in neurons, for example. Incertain instances, trisomy 12 has been identified in chronic lymphocyticleukemia (CLL) and trisomy 8 has been identified in acute myeloidleukemia (AML). Also, genetic syndromes in which an individual ispredisposed to breakage of chromosomes (chromosome instabilitysyndromes) are frequently associated with increased risk for varioustypes of cancer, thus highlighting the role of somatic aneuploidy incarcinogenesis. Methods and kits described herein can identify presenceor absence of non-mosaic and mosaic chromosome abnormalities.

Following is a non-limiting list of chromosome abnormalities that can bepotentially identified by methods and kits described herein.

Chromosome Abnormality Disease Association X XO Turner's Syndrome Y XXYKlinefelter syndrome Y XYY Double Y syndrome Y XXX Trisomy X syndrome YXXXX Four X syndrome Y Xp21 deletion Duchenne's/Becker syndrome,congenital adrenal hypoplasia, chronic granulomatus disease Y Xp22deletion steroid sulfatase deficiency Y Xq26 deletion X-linked lymphproliferative disease 1 1p (somatic) neuroblastoma monosomy trisomy 2monosomy trisomy growth retardation, developmental and mental delay, and2q minor physical abnormalities 3 monosomy trisomy Non-Hodgkin'slymphoma (somatic) 4 monosomy trsiomy Acute non lymphocytic leukemia(ANLL) (somatic) 5 5p Cri du chat; Lejeune syndrome 5 5q myelodysplasticsyndrome (somatic) monosomy trisomy 6 monosomy trisomy clear-cellsarcoma (somatic) 7 7q11.23 deletion William's syndrome 7 monosomytrisomy monosomy 7 syndrome of childhood; somatic: renal corticaladenomas; myelodysplastic syndrome 8 8q24.1 deletion Langer-Giedonsyndrome 8 monosomy trisomy myelodysplastic syndrome; Warkany syndrome;somatic: chronic myelogenous leukemia 9 monosomy 9p Alfi's syndrome 9monosomy 9p partial Rethore syndrome trisomy 9 trisomy complete trisomy9 syndrome; mosaic trisomy 9 syndrome 10 Monosomy trisomy ALL or ANLL(somatic) 11 11p- Aniridia; Wilms tumor 11 11q- Jacobson Syndrome 11monosomy (somatic) myeloid lineages affected (ANLL, MDS) trisomy 12monosomy trisomy CLL, Juvenile granulosa cell tumor (JGCT) (somatic) 1313q- 13q-syndrome; Orbeli syndrome 13 13q14 deletion retinoblastoma 13monosomy trisomy Patau's syndrome 14 monosomy trisomy myeloid disorders(MDS, ANLL, atypical CML) (somatic) 15 15q11-q13 deletion Prader-Willi,Angelman's syndrome monosomy 15 trisomy (somatic) myeloid and lymphoidlineages affected, e.g., MDS, ANLL, ALL, CLL) 16 16q13.3 deletionRubenstein-Taybi monosomy trisomy papillary renal cell carcinomas(malignant) (somatic) 17 17p-(somatic) 17p syndrome in myeloidmalignancies 17 17q11.2 deletion Smith-Magenis 17 17q13.3 Miller-Dieker17 monosomy trisomy renal cortical adenomas (somatic) 17 17p11.2-12trisomy Charcot-Marie Tooth Syndrome type 1; HNPP 18 18p- 18p partialmonosomy syndrome or Grouchy Lamy Thieffry syndrome 18 18q- Grouchy LamySalmon Landry Syndrome 18 monosomy trisomy Edwards Syndrome 19 monosomytrisomy 20 20p- trisomy 20p syndrome 20 20p11.2-12 deletion Alagille 2020q- somatic: MDS, ANLL, polycythemia vera, chronic neutrophilicleukemia 20 monosomy trisomy papillary renal cell carcinomas (malignant)(somatic) 21 monosomy trisomy Down's syndrome 22 22q11.2 deletionDiGeorge's syndrome, velocardiofacial syndrome, conotruncal anomaly facesyndrome, autosomal dominant Opitz G/BBB syndrome, Caylor cardiofacialsyndrome 22 monosomy trisomy complete trisomy 22 syndrome

In certain embodiments, presence or absence of a fetal chromosomeabnormality is identified (e.g., trisomy 21, trisomy 18 and/or trisomy13). In some embodiments, presence or absence of a chromosomeabnormality related to a cell proliferation condition or cancer isidentified. Presence or absence of one or more of the chromosomeabnormalities described in the table above may be identified in someembodiments.

In some embodiments, a prenatal or neonatal condition is a cellproliferation condition. Cell proliferation conditions include, withoutlimitation, cancers of the colorectum, breast, lung, liver, pancreas,lymph node, colon, prostate, brain, head and neck, skin, liver, kidney,and heart. Examples of cancers include hematopoietic neoplasticdisorders, which are diseases involving hyperplastic/neoplastic cells ofhematopoietic origin (e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof). The diseases can arise frompoorly differentiated acute leukemias, e.g., erythroblastic leukemia andacute megakaryoblastic leukemia. Additional myeloid disorders include,but are not limited to, acute promyeloid leukemia (APML), acutemyelogenous leukemia (AML) and chronic myelogenous leukemia (CML)(reviewed in Vaickus, Crit. Rev. in Oncol./Hemotol. 11:267-297 (1991));lymphoid malignancies include, but are not limited to acutelymphoblastic leukemia (ALL), which includes B-lineage ALL and T-lineageALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL),hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).Additional forms of malignant lymphomas include, but are not limited tonon-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas,adult T cell leukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL),large granular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease. In a particular embodiment, a cell proliferativedisorder is non-endocrine tumor or endocrine tumors. Illustrativeexamples of non-endocrine tumors include but are not limited toadenocarcinomas, acinar cell carcinomas, adenosquamous carcinomas, giantcell tumors, intraductal papillary mucinous neoplasms, mucinouscystadenocarcinomas, pancreatoblastomas, serous cystadenomas, solid andpseudopapillary tumors. An endocrine tumor may be an islet cell tumor.

Cell proliferative conditions also include inflammatory conditions, suchas inflammation conditions of the skin, including, for example, eczema,discoid lupus erythematosus, lichen planus, lichen sclerosus, mycosisfungoides, photodermatoses, pityriasis rosea, psoriasis. Also includedare cell proliferative conditions related to obesity, such asproliferation of adipocytes, for example.

Cell proliferative conditions also include viral diseases, including forexample, Acquired Immunodeficiency Syndrome, Adenoviridae Infections,Alphavirus Infections, Arbovirus Infections, Borna Disease, BunyaviridaeInfections, Caliciviridae Infections, Chickenpox, CoronaviridaeInfections, Coxsackievirus Infections, Cytomegalovirus Infections,Dengue, DNA Virus Infections, Ecthyma, Contagious, Encephalitis,Arbovirus, Epstein-Barr Virus Infections, Erythema Infectiosum,Hantavirus Infections, Hemorrhagic Fevers, Viral, Hepatitis, Viral,Human, Herpes Simplex, Herpes Zoster, Herpes Zoster Oticus,Herpesviridae Infections, Infectious Mononucleosis, Influenza in Birds,Influenza, Human, Lassa Fever, Measles, Molluscum Contagiosum, Mumps,Paramyxoviridae Infections, Phlebotomus Fever, Polyomavirus Infections,Rabies, Respiratory Syncytial Virus Infections, Rift Valley Fever, RNAVirus Infections, Rubella, Slow Virus Diseases, Smallpox, SubacuteSclerosing Panencephalitis, Tumor Virus Infections, Warts, West NileFever, Virus Diseases and Yellow Fever. For example, Large T antigen ofthe SV40 transforming virus acts on UBF, activates it and recruits otherviral proteins to Pol I complex, and thereby stimulates cellproliferation to promote virus propagation.

Cell proliferative conditions also include cardiac conditions resultingfrom cardiac stress, such as hypertension, balloon angioplasty, valvulardisease and myocardial infarction. For example, cardiomyocytes aredifferentiated muscle cells in the heart that constitute the bulk of theventricle wall, and vascular smooth muscle cells line blood vessels.Although both are muscle cell types, cardiomyocytes and vascular smoothmuscle cells vary in their mechanisms of contraction, growth anddifferentiation. Cardiomyocytes become terminally differentiated shortlyafter heart formation and thus lose the capacity to divide, whereasvascular smooth muscle cells are continually undergoing modulation fromthe contractile to proliferative phenotype. Under variouspathophysiological stresses such as hypertension, balloon angioplasty,valvular disease and myocardial infarction, for example, the heart andvessels undergo morphologic growth-related alterations that can reducecardiac function and eventually manifest in heart failure. Cellproliferative conditions also include conditions related to angiogenesis(e.g., cancers) and obesity caused by proliferation of adipocytes andother fat cells.

In some embodiments, methods and compositions described herein can beused to extract cell-free nucleic acids from biological samples, fromanimals or humans for example, for the purpose of detecting ordiagnosing a disease condition (e.g., cancer, genetic abnormality, andthe like). In certain embodiments, the biological sample is from ahuman, who also may be a cancer patient in certain embodiments. Methodsand compositions described herein may be used in conjunction with anymethod known to elevate nucleic acids (e.g., nucleotide sequences)associated with cancer conditions, from sample nucleic acid compositions(e.g., patient samples). Alternatively, methods and compositionsdescribed herein may be used in conjunction with any method known todecrease nucleic acid sequences associated with cancer conditions, fromin sample nucleotide compositions.

Nucleic Acids

Target or sample nucleic acid may be derived from one or more samples orsources. “Sample nucleic acid” as used herein refers to a nucleic acidfrom a sample. “Target nucleic acid” and “template nucleic acid” areused interchangeably throughout the document and refer to a nucleic acidof interest. The terms “total nucleic acid” or “nucleic acidcomposition” as used herein, refer to the entire population of nucleicacid species from or in a sample or source. Non-limiting examples ofnucleic acid compositions containing “total nucleic acids” include, hostand non-host nucleic acid, maternal and fetal nucleic acid, genomic andacellular nucleic acid, or mixed-population nucleic acids isolated fromenvironmental sources. As used herein, “nucleic acid” refers topolynucleotides such as deoxyribonucleic acid (DNA) and ribonucleic acid(RNA), and refers to derivatives, variants and analogs of RNA or DNAmade from nucleotide analogs, single (sense or antisense) anddouble-stranded polynucleotides. The term “nucleic acid” does not referto or infer a specific length of the polynucleotide chain, thusnucleotides, polynucleotides, and oligonucleotides are also includedwithin “nucleic acid.”

In some embodiments, target nucleic acid is relatively short and maycomprise fragments in the of about 5 to about 500 nucleotides or basepairs, for example. In certain embodiments, the target nucleic acid canbe in the range of about 5 to about 300 nucleotides or base pairs. Incertain embodiments, the relatively short target nucleic acid can be inthe range of about 5 to about 200 nucleotides or base pairs. That is,target nucleic acids can be about 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 210, 220, 230 250, 300, 350, 400, 450, or up toabout 500 nucleotides or base pairs in length. In certain embodiments,the relatively long nucleic acid can be greater than about 200nucleotides or base pairs. The term “nucleotides”, as used herein, inreference to the length of nucleic acid chain, refers to a singlestranded nucleic acid chain. The term “base pairs”, as used herein, inreference to the length of nucleic acid chain, refers to a doublestranded nucleic acid chain.

Deoxyribonucleotides include deoxyadenosine, deoxycytidine,deoxyguanosine and deoxythymidine. For RNA, the uracil base is uridine.A source or sample containing sample nucleic acid(s) may contain one ora plurality of sample nucleic acids. A plurality of sample nucleic acidsas described herein refers to at least 2 sample nucleic acids andincludes nucleic acid sequences that may be identical or different. Thatis, the sample nucleic acids may all be representative of the samenucleic acid sequence, or may be representative of two or more differentnucleic acid sequences (e.g., from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 50, 100, 1000 or more sequences).

A sample containing nucleic acids may be collected from an organism,mineral or geological site (e.g., soil, rock, mineral deposit, combattheater), forensic site (e.g., crime scene, contraband or suspectedcontraband), or a paleontological or archeological site (e.g., fossil,or bone) for example. A sample may be a “biological sample,” whichrefers to any material obtained from a living source or formerly-livingsource, for example, an animal such as a human or other mammal, a plant,a bacterium, a fungus, a protist or a virus. The biological sample canbe in any form, including without limitation a solid material such as atissue, cells, a cell pellet, a cell extract, or a biopsy, or abiological fluid such as urine, blood, saliva, amniotic fluid, exudatefrom a region of infection or inflammation, or a mouth wash containingbuccal cells, urine, cerebral spinal fluid and synovial fluid andorgans.

The biological sample can be maternal blood, including maternal plasmaor serum. In some circumstances, the biological sample is acellular. Inother circumstances, the biological sample does contain cellularelements or cellular remnants in maternal blood. Other biologicalsamples include amniotic fluid, chorionic villus sample, biopsy materialfrom a pre-implantation embryo, maternal urine, maternal saliva, acelocentesis sample, fetal nucleated cells or fetal cellular remnants,or the sample obtained from washings of the female reproductive tract.In some embodiments, a biological sample may be blood.

As used herein, the term “blood” encompasses whole blood or anyfractions of blood, such as serum and plasma as conventionally defined.Blood plasma refers to the fraction of whole blood resulting fromcentrifugation of blood treated with anticoagulants. Blood serum refersto the watery portion of fluid remaining after a blood sample hascoagulated. Fluid or tissue samples often are collected in accordancewith standard protocols hospitals or clinics generally follow. Forblood, an appropriate amount of peripheral blood (e.g., between 3-40milliliters) often is collected and can be stored according to standardprocedures prior to further preparation in such embodiments. A fluid ortissue sample from which template nucleic acid is extracted may beacellular. In some embodiments, a fluid or tissue sample may containcellular elements or cellular remnants. In some embodiments, the nucleicacid composition containing the target nucleic acid or nucleic acids maybe collected from a cell free or substantially cell free biologicalcomposition, blood plasma, blood serum or urine for example.

The term “substantially cell free” as used herein, refers tobiologically derived preparations or compositions that contain asubstantially small number of cells, or no cells. A preparation intendedto be completely cell free, but containing cells or cell debris can beconsidered substantially cell free. That is, substantially cell freebiological preparations can include up to about 50 cells or fewer permilliliter of preparation (e.g., up to about 50 cells per milliliter orless, 45 cells per milliliter or less, 40 cells per milliliter or less,35 cells per milliliter or less, 30 cells per milliliter or less, 25cells per milliliter or less, 20 cells per milliliter or less, 15 cellsper milliliter or less, 10 cells per milliliter or less, 5 cells permilliliter or less, or up to about 1 cell per milliliter or less).

For prenatal applications of technology described herein, fluid ortissue sample may be collected from a female at a gestational agesuitable for testing, or from a female who is being tested for possiblepregnancy. Suitable gestational age may vary depending on the chromosomeabnormality tested. In certain embodiments, a pregnant female subjectsometimes is in the first trimester of pregnancy, at times in the secondtrimester of pregnancy, or sometimes in the third trimester ofpregnancy. In certain embodiments, a fluid or tissue is collected from apregnant woman at 1-4, 4-8, 8-12, 12-16, 16-20, 20-24, 24-28, 28-32,32-36, 36-40, or 40-44 weeks of fetal gestation, and sometimes between5-28 weeks of fetal gestation.

Target and/or total nucleic acid can be extracellular nucleic acid incertain embodiments. The term “extracellular nucleic acid” as usedherein refers to nucleic acid isolated from a source havingsubstantially no cells (e.g., no detectable cells, or fewer than 50cells per milliliter or less as described above, or may contain cellularelements or cellular remnants). Examples of acellular sources forextracellular nucleic acid are blood plasma, blood serum and urine.Without being limited by theory, extracellular nucleic acid may be aproduct of cell apoptosis and cell breakdown, which provides basis forextracellular nucleic acid often having a series of lengths across alarge spectrum (e.g., a “ladder”). In some embodiments, the nucleicacids can be cell free nucleic acid.

Extracellular template nucleic acid can include different nucleic acidspecies. For example, blood serum or plasma from a person having cancercan include nucleic acid from cancer cells and nucleic acid fromnon-cancer cells. In another example, blood serum or plasma from apregnant female can include maternal nucleic acid and fetal nucleicacid. In some instances, fetal nucleic acid sometimes is about 5% toabout 40% of the overall template nucleic acid (e.g., about 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39% of the templatenucleic acid is fetal nucleic acid). In some embodiments, the majorityof fetal nucleic acid in template nucleic acid is of a length of about500 base pairs or less (e.g., about 80, 85, 90, 91, 92, 93, 94, 95, 96,97, 98, 99 or 100% of fetal nucleic acid is of a length of about 500base pairs or less).

The amount of fetal nucleic acid (e.g., concentration) in templatenucleic acid sometimes is determined. In certain embodiments, the amountof fetal nucleic acid is determined according to markers specific to amale fetus (e.g., Y-chromosome STR markers (e.g., DYS 19, DYS 385, DYS392 markers); RhD marker in RhD-negative females), or according to oneor more markers specific to fetal nucleic acid and not maternal nucleicacid (e.g., fetal RNA markers in maternal blood plasma; Lo, 2005,Journal of Histochemistry and Cytochemistry 53 (3): 293-296). The amountof fetal nucleic acid in extracellular template nucleic acid can bequantified and utilized for the identification of the presence orabsence of a chromosome abnormality in certain embodiments.

In some embodiments, extracellular nucleic acid can be enriched orrelatively enriched for fetal nucleic acid, using methods describedherein alone, or in conjunction with other methods known in the art.Non-limiting examples of additional methods known in the art forenriching a sample for a particular species of nucleic acid aredescribed in; PCT Patent Application Number PCT/US07/69991, filed May30, 2007, PCT Patent Application Number PCT/US2007/071232, filed Jun.15, 2007, U.S. Provisional Application Nos. 60/968,876 and 60/968,878,and PCT Patent Application Number PCT/EP05/012707, filed Nov. 28, 2005,herein incorporated by reference in their entirety. In certainembodiments, maternal nucleic acid can be selectively removed(partially, substantially, almost completely or completely) from thesample.

A sample also may be isolated at a different time point as compared toanother sample, where each of the samples may be from the same or adifferent source. A sample nucleic acid may be from a nucleic acidlibrary, such as a cDNA or RNA library, for example. A sample nucleicacid may be a result of nucleic acid purification or isolation and/oramplification of nucleic acid molecules from the sample. Sample nucleicacid provided for sequence analysis processes described herein maycontain nucleic acid from one sample or from two or more samples (e.g.,from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or 20 samples).

Sample nucleic acid may comprise or consist essentially of any type ofnucleic acid suitable for use with processes of the technology, such assample nucleic acid that can hybridize to solid phase nucleic acid(described hereafter), for example. A sample nucleic in certainembodiments can comprise or consist essentially of DNA (e.g.,complementary DNA (cDNA), genomic DNA (gDNA) and the like), RNA (e.g.,message RNA (mRNA), short inhibitory RNA (siRNA), microRNA, ribosomalRNA (rRNA), tRNA and the like), and/or DNA or RNA analogs (e.g.,containing base analogs, sugar analogs and/or a non-native backbone andthe like). A nucleic acid can be in any form useful for conductingprocesses herein (e.g., linear, circular, supercoiled, single-stranded,double-stranded and the like). A nucleic acid may be, or may be from, aplasmid, phage, autonomously replicating sequence (ARS), centromere,artificial chromosome, chromosome, a cell, a cell nucleus or cytoplasmof a cell in certain embodiments. A sample nucleic acid in someembodiments is from a single chromosome (e.g., a nucleic acid sample maybe from one chromosome of a sample obtained from a diploid organism).

Sample nucleic acid may be provided for conducting methods describedherein without processing of the sample(s) containing the nucleic acidin certain embodiments. In some embodiments, sample nucleic acid isprovided for conducting methods described herein after processing of thesample(s) containing the nucleic acid. For example, a sample nucleicacid may be extracted, isolated, purified or amplified from thesample(s). The term “isolated” as used herein refers to nucleic acidremoved from its original environment (e.g., the natural environment ifit is naturally occurring, or a host cell if expressed exogenously), andthus is altered “by the hand of man” from its original environment. Anisolated nucleic acid generally is provided with fewer non-nucleic acidcomponents (e.g., protein, lipid) than the amount of components presentin a source sample. A composition comprising isolated sample nucleicacid can be substantially isolated (e.g., about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or greater than 99% free of non-nucleic acidcomponents). The term “purified” as used herein refers to sample nucleicacid provided that contains fewer nucleic acid species than in thesample source from which the sample nucleic acid is derived. Acomposition comprising sample nucleic acid may be substantially purified(e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greaterthan 99% free of other nucleic acid species). The term “amplified” asused herein refers to subjecting nucleic acid of a sample to a processthat linearly or exponentially generates amplicon nucleic acids havingthe same or substantially the same nucleotide sequence as the nucleotidesequence of the nucleic acid in the sample, or portion thereof.

Sample nucleic acid also may be processed by subjecting nucleic acid toa method that generates nucleic acid fragments, in certain embodiments,before providing sample nucleic acid for a process described herein. Insome embodiments, sample nucleic acid subjected to fragmentation orcleavage may have a nominal, average or mean length of about 5 to about10,000 base pairs, about 100 to about 1,000 base pairs, about 100 toabout 500 base pairs, or about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10000 basepairs. Fragments can be generated by any suitable method known in theart, and the average, mean or nominal length of nucleic acid fragmentscan be controlled by selecting an appropriate fragment-generatingprocedure by the person of ordinary skill. In certain embodiments,sample nucleic acid of a relatively shorter length can be utilized toanalyze sequences that contain little sequence variation and/or containrelatively large amounts of known nucleotide sequence information. Insome embodiments, sample nucleic acid of a relatively longer length canbe utilized to analyze sequences that contain greater sequence variationand/or contain relatively small amounts of unknown nucleotide sequenceinformation.

Sample nucleic acid fragments often contain overlapping nucleotidesequences, and such overlapping sequences can facilitate construction ofa nucleotide sequence of the previously non-fragmented sample nucleicacid, or a portion thereof. For example, one fragment may havesubsequences x and y and another fragment may have subsequences y and z,where x, y and z are nucleotide sequences that can be 5 nucleotides inlength or greater. Overlap sequence y can be utilized to facilitateconstruction of the x-y-z nucleotide sequence in nucleic acid from asample. Sample nucleic acid may be partially fragmented (e.g., from anincomplete or terminated specific cleavage reaction) or fully fragmentedin certain embodiments.

Sample nucleic acid can be fragmented by various methods known to theperson of ordinary skill, which include without limitation, physical,chemical and enzymic processes. Examples of such processes are describedin U.S. Patent Application Publication No. 20050112590 (published on May26, 2005, entitled “Fragmentation-based methods and systems for sequencevariation detection and discovery,” naming Van Den Boom et al.). Certainprocesses can be selected by the person of ordinary skill to generatenon-specifically cleaved fragments or specifically cleaved fragments.Examples of processes that can generate non-specifically cleavedfragment sample nucleic acid include, without limitation, contactingsample nucleic acid with apparatus that expose nucleic acid to shearingforce (e.g., passing nucleic acid through a syringe needle; use of aFrench press); exposing sample nucleic acid to irradiation (e.g., gamma,x-ray, UV irradiation; fragment sizes can be controlled by irradiationintensity); boiling nucleic acid in water (e.g., yields about 500 basepair fragments) and exposing nucleic acid to an acid and base hydrolysisprocess.

Sample nucleic acid may be specifically cleaved by contacting thenucleic acid with one or more specific cleavage agents. The term“specific cleavage agent” as used herein refers to an agent, sometimes achemical or an enzyme that can cleave a nucleic acid at one or morespecific sites. Specific cleavage agents often will cleave specificallyaccording to a particular nucleotide sequence at a particular site.

Examples of enzymic specific cleavage agents include without limitationendonucleases (e.g., DNase (e.g., DNase I, II); RNase (e.g., RNase E, F,H, P); Cleavase™ enzyme; Taq DNA polymerase; E. coli DNA polymerase Iand eukaryotic structure-specific endonucleases; murine FEN-1endonucleases; type I, II or III restriction endonucleases such as AccI, Afl III, Alu I, Alw44 I, Apa I, Asn I, Ava I, Ava II, BamH I, Ban II,Bcl I, Bgl I. Bgl II, Bln I, Bsm I, BssH II, BstE II, Cfo I, Cla I, DdeI, Dpn I, Dra I, EcIX I, EcoR I, EcoR I, EcoR II, EcoR V, Hae II, HaeII, Hind II, Hind III, Hpa I, Hpa II, Kpn I, Ksp I, Mlu I, MIuN I, MspI, Nci I, Nco I, Nde I, Nde II, Nhe I, Not I, Nru I, Nsi I, Pst I, PvuI, Pvu II, Rsa I, Sac I, Sal I, Sau3A I, Sca I, ScrF I, Sfi I, Sma I,Spe I, Sph I, Ssp I, Stu I, Sty I, Swa I, Taq I, Xba I, Xho I.);glycosylases (e.g., uracil-DNA glycolsylase (UDG), 3-methyladenine DNAglycosylase, 3-methyladenine DNA glycosylase II, pyrimidine hydrate-DNAglycosylase, FaPy-DNA glycosylase, thymine mismatch-DNA glycosylase,hypoxanthine-DNA glycosylase, 5-Hydroxymethyluracil DNA glycosylase(HmUDG), 5-Hydroxymethylcytosine DNA glycosylase, or 1,N6-etheno-adenineDNA glycosylase); exonucleases (e.g., exonuclease III); ribozymes, andDNAzymes. Sample nucleic acid may be treated with a chemical agent, orsynthesized using modified nucleotides, and the modified nucleic acidmay be cleaved. In non-limiting examples, sample nucleic acid may betreated with (i) alkylating agents such as methylnitrosourea thatgenerate several alkylated bases, including N3-methyladenine andN3-methylguanine, which are recognized and cleaved by alkyl purineDNA-glycosylase; (ii) sodium bisulfite, which causes deamination ofcytosine residues in DNA to form uracil residues that can be cleaved byuracil N-glycosylase; and (iii) a chemical agent that converts guanineto its oxidized form, 8-hydroxyguanine, which can be cleaved byformamidopyrimidine DNA N-glycosylase. Examples of chemical cleavageprocesses include without limitation alkylation, (e.g., alkylation ofphosphorothioate-modified nucleic acid); cleavage of acid lability ofP3′-N5′-phosphoroamidate-containing nucleic acid; and osmium tetroxideand piperidine treatment of nucleic acid.

As used herein, the term “complementary cleavage reactions” refers tocleavage reactions that are carried out on the same sample nucleic acidusing different cleavage reagents or by altering the cleavagespecificity of the same cleavage reagent such that alternate cleavagepatterns of the same target or reference nucleic acid or protein aregenerated. In certain embodiments, sample nucleic acid may be treatedwith one or more specific cleavage agents (e.g., 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more specific cleavage agents) in one or more reaction vessels(e.g., sample nucleic acid is treated with each specific cleavage agentin a separate vessel).

Sample nucleic acid also may be exposed to a process that modifiescertain nucleotides in the nucleic acid before providing sample nucleicacid for a method described herein. A process that selectively modifiesnucleic acid based upon the methylation state of nucleotides therein canbe applied to sample nucleic acid. The term “methylation state” as usedherein refers to whether a particular nucleotide in a polynucleotidesequence is methylated or not methylated. Methods for modifying a targetnucleic acid molecule in a manner that reflects the methylation patternof the target nucleic acid molecule are known in the art, as exemplifiedin U.S. Pat. No. 5,786,146 and U.S. patent publications 20030180779 and20030082600. For example, non-methylated cytosine nucleotides in anucleic acid can be converted to uracil by bisulfite treatment, whichdoes not modify methylated cytosine. Non-limiting examples of agentsthat can modify a nucleotide sequence of a nucleic acid includemethylmethane sulfonate, ethylmethane sulfonate, diethylsulfate,nitrosoguanidine (N-methyl-N′-nitro-N-nitrosoguanidine), nitrous acid,di-(2-chloroethyl)sulfide, di-(2-chloroethyl)methylamine, 2-aminopurine,t-bromouracil, hydroxylamine, sodium bisulfite, hydrazine, formic acid,sodium nitrite, and 5-methylcytosine DNA glycosylase. In addition,conditions such as high temperature, ultraviolet radiation, x-radiation,can induce changes in the sequence of a nucleic acid molecule.

Sample nucleic acid may be provided in any form useful for conducting asequence analysis or manufacture process described herein, such as solidor liquid form, for example. In certain embodiments, sample nucleic acidmay be provided in a liquid form optionally comprising one or more othercomponents, including without limitation one or more buffers or saltsselected by the person of ordinary skill.

Solid Supports

The term “solid support” or “solid phase” as used herein refers to aninsoluble material with which nucleic acid can be associated, and theterms can be used interchangeably. Examples of solid supports for usewith processes described herein include, without limitation, chips, flatsurfaces filters, one or more capillaries and/or fibers, arrays,filters, beads, beads (e.g., paramagnetic beads, magnetic beads,microbeads, nanobeads) and particles (e.g., microparticles,nanoparticles). Beads and/or particles may be free or in connection withone another (e.g., sintered). In some embodiments, the solid phase canbe a collection of particles. In certain embodiments, the particles cancomprise silica, and the silica may comprise silica dioxide. In someembodiments the silica can be porous, and in certain embodiments thesilica can be non-porous. In some embodiments, the particles furthercomprise an agent that confers a paramagnetic property to the particles.In certain embodiments, the agent comprises a metal, and in certainembodiments the agent is a metal oxide, (e.g., iron or iron oxides,where the iron oxide contains a mixture of Fe2+ and Fe3+). Magneticallyresponsive silica dioxide beads can be obtained commercially.Non-limiting examples of magnetically responsive silica beads are;DynaL® beads (Invitrogen, Carlsbad, Calif.), SiMag® beads (Chemicell,Berlin, Germany), MagAttract® beads (Qiagen, Hilden, Germany), Magnesil®beads (Promega, Madison, Wis.), and functional magnetic silica beads(MoBiTec, Gottingen, Germany; Microspheres-Nanospheres.com (a divisionof Corpuscular, Inc) Lincolndale, N.Y.; G. Kisker Biotech, Steinfurt,Germany).

In some embodiments, the solid phase does not comprise a functionalgroup that interacts with the nucleic acid. In certain embodiments, thesolid phase does not comprise a carboxy functional group. In someembodiments, the solid phase has a net charge. In certain embodiments,the net charge is positive, and sometimes the net charge is negative.

Nucleic acids may reversibly associate with a solid support (e.g.,magnetic silica dioxide particles) under association conditions. Theassociation may be reversed under dissociation conditions, and all or asubset of nucleic acid associated with the solid phase may dissociatefrom the solid phase under the dissociation conditions. The term“associate” as used herein refers to an interaction between a nucleicacid and a solid phase, which interaction often is non-covalent, oftenis adsorption, sometimes is absorption, often is binding, and generallyis reversible. The term “association conditions” as used herein, refersto conditions under which nucleic acid from a nucleic acid compositionis associated with a solid support. In some embodiments, nucleic acid ofsubstantially all sizes in the composition associates with a solidsupport under the association conditions. Sometimes, substantially allof the nucleic acid in a composition associates with a solid support,and sometimes about 30 percent to about 100 percent of the nucleic acid,from the total nucleic acid in a sample, associates or binds to thesolid support (e.g., 30% or greater, 35% or greater, 40% or greater, 45%or greater, 50% or greater, 55% or greater, 60% or greater, 65% orgreater, 70% or greater, 75% or greater, 80% or greater, 85% or greater,90% or greater, 95% or greater, or 99% or greater of the total nucleicacid present in a sample associates with the solid phase).

In some embodiments, association conditions can include one or more ofthe following: salts, alcohols, volume excluding agents (e.g., sometimesalso referred to as crowding agents), or combinations thereof. Salts maycomprise chaotropic salts, ionic salts or a combination of such salts.Non-limiting examples of chaotropic salts include guanidine salt,guanidinium salt, sodium iodide, potassium iodide, sodium thiocyanateand urea. Non-limiting examples of ionic salts include sodium chloride,magnesium chloride, calcium chloride, potassium chloride, lithiumchloride, barium chloride, cesium chloride, ammonium acetate, sodiumacetate, ammonium perchlorate and sodium perchlorate. In someembodiments, a chaotropic salt can be a guanidine salt (e.g., guanidine(iso)thiocyanate, for example). In certain embodiments, an ionic saltcan be a sodium salt (e.g., sodium chloride, for example).

In certain embodiments, a salt may be introduced at a concentrationsufficient to associate the nucleic acid to a solid support (e.g.,substantially all of the nucleic acid), and the salt may be the onlycomponent that associates the nucleic acid to the solid phase or more beutilized in combination with other components to perform the samefunction. Salt concentrations for binding nucleic acids may be dependenton length of nucleic acid, base sequence, combinations thereof and thelike, and can be determined. In some embodiments a salt is utilized inan amount that yields a final salt concentration in the range of about0.25M to about 5M of the salt (e.g., 0.5M, 1M, 1.5M, 2M, 2.5M, 3M, 4M,or 5M). Salt concentrations also can be expressed as percent weight pervolume and salt concentration ranges expressed as ranges of percentweight per volume (e.g., 40 to 60% weight per volume), and can be usedinterchangeably with Molar concentrations. In some embodiments, the saltconcentration chosen may be sufficient to bind substantially allnon-target nucleic acid to a solid support, while minimizing the bindingof target nucleic acid. In certain embodiments, the salt concentrationchosen may be sufficient to associate all or substantially all thenucleic acid in solution. In some embodiments a salt can be added toyield a solution with a concentration in the range of about 5% to about60% weight per volume that may be sufficient to associate target ortotal nucleic acid to a solid support. In some embodiments, additionalsolid phase also may be used to ensure capture of all nucleic acid froma sample.

Alcohols suitable for use in association conditions with the methodsdescribed herein are the C1-C6 alkyl alcohols, and their branched chainderivatives or isoforms. Non-limiting examples of the C1-06 alcohols aremethanol (C1), ethanol (C2), propanol (C3), butanol (C4), pentanol (C5),and hexanol (C6), and linear and branched variants thereof. In someembodiments the alkyl alcohol is included in a final amount (percentvolume of alcohol in water or aqueous buffered solution) in the range ofabout 25% or more, 30% or more, 35% or more, 40% or more, 45% or more,50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% ormore, 80% or more, 85% or more, 90% or more, 95% or more, or up to 99%or more. In some embodiments, ethanol is used for associating nucleicacids with the solid phase (e.g., magnetically responsive silica dioxidebeads). In certain embodiments, the final concentration of ethanol isabout 33%. In some embodiments, an alcohol is used as a wash solution toremove impurities. In embodiments using an alcohol as a wash solution,the alcohol often is between about 75% to about 95% alcohol (e.g.,ethanol).

Volume excluding agents sometimes may be included in associationconditions, in some embodiments. In certain embodiments, volumeexcluding agents can be used in size selection (e.g., dissociation)buffers or solutions. Volume excluding agents can be suitable for use in(i) association conditions, and/or (ii) dissociation conditions thatallow for preferential dissociation of nucleic acid of a particular size(e.g., size selection). Volume excluding agents include, withoutlimitation, polyalkyl glycol (e.g., polyethylene glycol (PEG), forexample), dextran, Ficoll, polyvinyl pyrollidone or combinationsthereof. In some embodiments, volume exclusion agents (also referred toas “crowding agents”) can be added to yield a solution containingbetween about 5% to about 30% volume exclusion agent, and morespecifically between about 8% to about 20% volume exclusion agent. Thatis, a volume excluding agent may be added to size selection ordissociation conditions to yield solutions containing up to about 5%volume excluding agent, up to about 6%, up to about 7%, up to about 8%,up to about 9%, up to about 10%, up to about 11%, up to about 12%, up toabout 13%, up to about 14%, up to about 15%, up to about 16%, up toabout 17%, up to about 18%, up to about 19%, up to about 20%, up toabout 25%, up to about, and up to about 30% volume excluding agent.

The term “dissociation conditions” as used herein refers to conditionsunder which (i) a subset of nucleic acid associated with the solidphase, or (ii) substantially all of the nucleic acid associated with asolid phase, is removed from the solid phase. For example, targetnucleic acid may exist in a population that is smaller than 300nucleotides or base pairs, and dissociation conditions may be selectedto selectively dissociate nucleic acids smaller than 300 nucleotides orbase pairs. The terms “preferential dissociation”, “preferentiallydissociates” and grammatical variants thereof, as used herein, refers toconditions under which target nucleic acids within a specific size range(e.g., between 5 and 300 nucleotides or base pairs, for example) aresubstantially or completely eluted from the solid support, while thelarger, non-target nucleic acid remains substantially bound. That is, aspecifically selected size range of nucleic acids (e.g., relativelyshort nucleic acids) may be preferentially removed from the solidsupport under the appropriate dissociation conditions, while leavingbehind the larger, unwanted or non-target nucleic acids.

The term “eluate” as used herein refers to the solution portion in acomposition that comprises a solid phase and a solution. An eluate underdissociation conditions can include relatively short nucleic acid andrelatively long nucleic acid dissociated from the solid phase, where therelatively short nucleic acid is preferentially dissociated from thesolid phase as compared to the relatively long nucleic acid under thedissociation conditions, in some embodiments. Thus, in certainembodiments, an eluate can include about 1.5-fold to about 5-fold morerelatively short nucleic acid as compared to relatively long nucleicacid, where the relatively short nucleic acid is about 300 base pairs orless (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 210, 220, 230, 340, 250, 260, 270, 280,290 base pairs), after the nucleic acid and solid phase have beenexposed to dissociation conditions to completion.

Certain dissociation conditions for eluting specific size ranges ofnucleic acids are presented below in Table 1 (presented below in Example3). In some embodiments, dissociation conditions contain one or moresalts (e.g., ionic salt, chaotropic salt) and one or more volumeexclusion agents (e.g., polyalkyl glycol, Ficoll, dextran, polyvinylpyrollidone (PVP) and the like). In some embodiments, a dissociationcondition may include C1-C6 alkyl alcohols. The components utilized inthe dissociation conditions can be utilized in any suitable amount thatallow for preferential dissociation of a relatively short nucleic acid,in some embodiments. In some embodiments, an ionic salt may be utilizedin dissociation conditions in an amount between about 0.05M to about2.0M, and sometimes between about 0.05M to about 1.0M (e.g., about 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 M). In some embodiments,the ionic salt used for dissociation of specific size fractions ofnucleic acids is sodium chloride (NaCl). In some embodiments theconcentration of sodium chloride is in the range of about 0.25M to about1.0M NaCl, and specific concentrations useful for isolating specificsized fractions may be found in Table 1.

In certain embodiments, a volume exclusion agent may be utilized indissociation conditions in an amount between about 5% to about 30%(e.g., weight to volume). Where the volume exclusion agent is apolyalkyl glycol, the polyalkyl glycol sometimes is utilized within arange of about 5% to about 25% in certain embodiments (e.g., about 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20%). The polyalkylglycol can have an average, mean or nominal molecular weight of about10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000,20000, 30000, 40000, 50000, 60000, 70000, 80000 or 90000 grams per mole.The polyalkyl glycol sometimes is branched or linear, and sometimes is apolyethylene glycol (PEG).

Where one volume exclusion agent is utilized at an optimum amount, theamount of a different volume exclusion agent for alternativedissociation conditions can be optimized based in part on the molecularweight of the different volume exclusion agent. For example, if a volumeexclusion agent that has been included in optimized dissociationconditions has a particular molecular weight, a different volumeexclusion agent having a higher molecular weight sometimes will beutilized at a lower amount, and a different volume exclusion agenthaving a lower molecular weight sometimes will be utilized at a higheramount. Where PEG8000 is utilized at a particular percentage foroptimized dissociation conditions, for example, a person of ordinaryskill in the art often use a lower amount of a different volumeexclusion agent having a higher molecular weight (e.g., PEG16000,Ficoll, dextran or polyvinyl pyrollidone), and often will use a higheramount of a different volume exclusion agent having a lower molecularweight (e.g., PEG4000). In some embodiments, dissociation conditions caninclude about 5% to about 8% Ficoll, about 2% to about 4% dextran, orabout 8% to about 10% polyvinyl pyrollidone (PVP). The PVP sometimes isPVP40 and often is not PVP10.

Particles or beads having a nominal, average or mean diameter of about 1nanometer to about 500 micrometers can be utilized, such as those havinga nominal, mean or average diameter, for example, of about 10 nanometersto about 100 micrometers; about 100 nanometers to about 100 micrometers;about 1 micrometer to about 100 micrometers; about 10 micrometers toabout 50 micrometers; about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700,800 or 900 nanometers; or about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500micrometers.

A solid support can comprise virtually any insoluble or solid material,and often a solid support composition is selected that is insoluble inwater. For example, a solid support can comprise or consist essentiallyof silica gel, glass (e.g. controlled-pore glass (CPG)), nylon,Sephadex®, Sepharose®, cellulose, a metal surface (e.g. steel, gold,silver, aluminum, silicon and copper), a magnetic material, a plasticmaterial (e.g., polyethylene, polypropylene, polyamide, polyester,polyvinylidenedifluoride (PVDF)) and the like. Beads or particles may beswellable (e.g., polymeric beads such as Wang resin) or non-swellable(e.g., CPG). Commercially available examples of beads include withoutlimitation Wang resin, Merrifield resin and Dynabeads® and SoluLink.

A solid support may be provided in a collection of solid supports. Asolid support collection comprises two or more different solid supportspecies. The term “solid support species” as used herein refers to asolid support in association with one particular solid phase nucleicacid species or a particular combination of different solid phasenucleic acid species. In certain embodiments, a solid support collectioncomprises 2 to 10,000 solid support species, 10 to 1,000 solid supportspecies or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600,700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or10000 unique solid support species. The solid supports (e.g., beads) inthe collection of solid supports may be homogeneous (e.g., all are Wangresin beads) or heterogeneous (e.g., some are Wang resin beads and someare magnetic beads). Each solid support species in a collection of solidsupports sometimes is labeled with a specific identification tag. Anidentification tag for a particular solid support species sometimes is anucleic acid (e.g., “solid phase nucleic acid”) having a unique sequencein certain embodiments. An identification tag can be any molecule thatis detectable and distinguishable from identification tags on othersolid support species.

In certain embodiments, a biological sample can be contacted with asolid support in the presence of a lysing/binding reagent to bind allnucleic acids to a solid support, the inhibitors are washed away, thensize selection is performed by adding different concentrations of saltsand crowding agents to the solid support selectively removing smallerfragments and leaving larger fragments on the solid support. Additionalsize selected elutions can be performed if a particular range offragments is required that is not eluted in the first size elution.Larger fragments or non-target fragments also can be enriched by elutingthe larger fragments from the solid support under appropriateconditions, or by eluting the smaller fragments and removing thesupernatant to a new tube, where the larger fragments remain on thesolid support and are thereby enriched. In some embodiments, the elutedsmall or target fragments can be further concentrated, enriched and/orpurified by binding to new beads in the presence of the appropriateconcentration of salt and precipitating agent, washing to removenon-nucleic acid impurities, and eluting in an appropriate aqueousbuffer or water.

Enrichment

Target nucleic acid (e.g., relatively short nucleic acids from about 50to about 200 nucleotides or base pairs in length), can be enrichedrelative to the target nucleic acid concentration in a total nucleicacid composition, or with respect to larger non-target nucleic acidfractions, using methods and compositions described herein. In someembodiments, relatively short nucleic acids may be enriched relative tothe total population of nucleic acids from a sample. Total nucleic acidfrom a sample may be bound to solid support under appropriateassociation conditions (see Example 1 for a non-limiting example ofappropriate association conditions). Relatively short, target nucleicacids may be purified by collecting the solid support (e.g., bycentrifugation or use of a magnetic field for paramagnetic particles,for example), and optionally removing the supernatant to a new tube,after incubating under dissociation conditions for a sufficient periodof time that preferentially release the relatively short nucleic acidfrom the solid phase. The relatively short nucleic acid is therebyenriched, relative to total nucleic acid by virtue of preferentialdissociation from the solid phase relative to the relatively largenon-target nucleic acid.

In certain embodiments, enrichment is a measure of the percent increasein the amount of relatively short nucleic acid in the disassociatednucleic acid as compared to in the nucleic acid composition subjected tothe enrichment process (e.g., percent increase in the relatively smallnucleic acid). In certain embodiments, this measure of enrichment isabout 10% to about 45% (e.g., about 15, 20, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44% enrichment).

In some embodiments, enrichment is a ratio of relatively small nucleicacid to relatively large nucleic acid in all of the nucleic acid elutedfrom the solid support under dissociation conditions. In certainembodiments, the ratio is about 1.05 to about 5 (e.g., ratio of about1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8,4.0, 4.2, 4.4, 4.6, 4.8 relatively short nucleic acid to relatively longnucleic acid).

Further enrichment of subspecies of relatively short target nucleicacids also may be performed using similar procedures and the appropriateassociation and dissociation conditions, using the size selected nucleicacids described above, in certain embodiments. Longer target, ornon-target nucleic acids also may be enriched using similar methods. Thetotal nucleic acid can be subjected to association conditions sufficientto bind only larger nucleic acids, while leaving smaller nucleic acidsin solution. The solid support is removed from the non-bound nucleicacids in the supernatant, thereby enriching larger nucleic acids.

Enriched nucleic acids of any size also may be further concentratedusing the methods and compositions described herein. Concentration ofnucleic acids may be performed by binding the size selected fraction ofnucleic acids, under appropriate association conditions, washing, one ormore times, to remove impurities, followed by dissociation (elution) ina smaller volume, of an appropriate buffer or solution, than theoriginal starting volume, thereby concentrating the previously sizeselected fraction. In some embodiments, concentration and additionalsize selection can be performed concurrently using the appropriateelution or dissociation buffer, as shown in Table 1 (see Example 3).Concentration also may be achieved by precipitating dissociated targetnucleic, for example.

FIG. 2 (see Example 2) illustrates the successful enrichment ofrelatively short nucleic acids in relation to the relatively longnucleic acids. The fold enrichment is calculated to be approximately 30%enrichment (e.g., 100%−(9%/13%)) of the male fetal DNA, and is achievedby selecting for nucleic acids 300 nucleotides or base pairs and lower.

Amplification

In some embodiments, it may be desirable to amplify the target sequenceusing any of several nucleic acid amplification procedures (described ingreater detail below). Nucleic acid amplification may be particularlybeneficial when target sequences exist at low copy number, or the targetsequences are non-host sequences and represent a small portion of thetotal nucleic acid in the sample (e.g., fetal nucleic acid in a maternalnucleic acid background). In some embodiments, amplification of targetsequences may aid in detection of gene dosage imbalances, as might beseen in genetic disorders involving chromosomal aneuploidy, for example.In some embodiments it may be desirable to amplify target nucleic acidsthat have been size selected using methods and compositions describedherein. In certain embodiments, total nucleic acid isolated fromsubstantially cell free samples may be amplified prior to using sizeselection methods and compositions described herein. In someembodiments, size selection of nucleic acid species of a particular sizerange (e.g., between about 50 to about 300 nucleotides of base pairs, orbetween about 50 to about 200 nucleotides or base pairs, for example)can be performed prior to amplification, to allow amplification andfurther enrichment of only target nucleic acid species. Nucleic acidamplification often involves enzymatic synthesis of nucleic acidamplicons (copies), which contain a sequence complementary to anucleotide sequence species being amplified. An amplification product(amplicon) of a particular nucleotide sequence species (e.g., targetsequence) is referred to herein as an “amplified nucleic acid species.”Amplifying target sequences and detecting the amplicon synthesized, canimprove the sensitivity of an assay, since fewer target sequences areneeded at the beginning of the assay, and can improve detection oftarget sequences.

The terms “amplify”, “amplification”, “amplification reaction”, or“amplifying” refers to any in vitro processes for multiplying the copiesof a target sequence of nucleic acid. Amplification sometimes refers toan “exponential” increase in target nucleic acid. However, “amplifying”as used herein can also refer to linear increases in the numbers of aselect target sequence of nucleic acid, but is different than aone-time, single primer extension step. In some embodiments, a one-time,single oligonucleotide extension step can be used to generate a doublestranded nucleic acid feature (e.g., synthesize the complement of arestriction endonuclease cleavage site contained in a single strandedoligonucleotide species, thereby creating a restriction site).

In some embodiments, a limited amplification reaction, also known aspre-amplification, can be performed. Pre-amplification is a method inwhich a limited amount of amplification occurs due to a small number ofcycles, for example 10 cycles, being performed. Pre-amplification canallow some amplification, but stops amplification prior to theexponential phase, and typically produces about 500 copies of thedesired nucleotide sequence(s). Use of pre-amplification may also limitinaccuracies associated with depleted reactants in standard PCRreactions, and also may reduce amplification biases due to nucleotidesequence or species abundance of the target. In some embodiments, aone-time primer extension may be used may be performed as a prelude tolinear or exponential amplification. In some embodiments, amplificationof the target nucleic acid may not be required, due to the use of ultrasensitive detections methods (e.g., single nucleotide sequencing,sequencing by synthesis and the like).

Where amplification may be desired, any suitable amplification techniquecan be utilized. Non-limiting examples of methods for amplification ofpolynucleotides include, polymerase chain reaction (PCR); ligationamplification (or ligase chain reaction (LCR)); amplification methodsbased on the use of Q-beta replicase or template-dependent polymerase(see US Patent Publication Number US20050287592); helicase-dependantisothermal amplification (Vincent et al., “Helicase-dependent isothermalDNA amplification”. EMBO reports 5 (8): 795-800 (2004)); stranddisplacement amplification (SDA); thermophilic SDA nucleic acid sequencebased amplification (3SR or NASBA) and transcription-associatedamplification (TAA). Non-limiting examples of PCR amplification methodsinclude standard PCR, AFLP-PCR, Allele-specific PCR, Alu-PCR, AsymmetricPCR, Colony PCR, Hot start PCR, Inverse PCR (IPCR), In situ PCR (ISH),Intersequence-specific PCR (ISSR-PCR), Long PCR, Multiplex PCR, NestedPCR, Quantitative PCR, Reverse Transcriptase PCR (RT-PCR), Real TimePCR, Single cell PCR, Solid phase PCR, combinations thereof, and thelike. Reagents and hardware for conducting PCR are commerciallyavailable.

In some embodiments, amplification target nucleic acid may beaccomplished by any suitable method available to one of skill in the artor selected from the listing above (e.g., ligase chain reaction (LCR),transcription-mediated amplification, and self-sustained sequencereplication or nucleic acid sequence-based amplification (NASBA)). Morerecently developed branched-DNA technology also may be used to amplifythe signal of target nucleic acids. For a review of branched-DNA (bDNA)signal amplification for direct quantification of nucleic acid sequencesin clinical samples, see Nolte, Adv. Clin. Chem. 33:201-235, 1998.

Amplification also can be accomplished using digital PCR, in certainembodiments (e.g., Kalinina and colleagues (Kalinina et al., “Nanoliterscale PCR with TaqMan detection.” Nucleic Acids Research. 25; 1999-2004,(1997); Vogelstein and Kinzler (Digital PCR. Proc Natl Acad Sci USA. 96;9236-41, (1999); PCT Patent Publication No. WO05023091A2 (incorporatedherein in its entirety); US Patent Publication No. 20070202525(incorporated herein in its entirety)). Digital PCR takes advantage ofnucleic acid (DNA, cDNA or RNA) amplification on a single moleculelevel, and offers a highly sensitive method for quantifying low copynumber nucleic acid. Systems for digital amplification and analysis ofnucleic acids are available (e.g., Fluidigm® Corporation).

In some embodiments, where RNA nucleic acid species may be used fordetection of fetal sequences, a DNA copy (cDNA) of the RNA transcriptsof interest can be synthesized prior to the amplification step. The cDNAcopy can be synthesized by reverse transcription, which may be carriedout as a separate step, or in a homogeneous reversetranscription-polymerase chain reaction (RT-PCR), a modification of thepolymerase chain reaction for amplifying RNA. Methods suitable for PCRamplification of ribonucleic acids are described by Romero and Rotbartin Diagnostic Molecular Biology: Principles and Applications pp.401-406; Persing et al., eds., Mayo Foundation, Rochester, Minn., 1993;Egger et al., J. Clin. Microbiol. 33:1442-1447, 1995; and U.S. Pat. No.5,075,212.

Use of a primer extension reaction also can be applied in methods of thetechnology. A primer extension reaction operates, for example, bydiscriminating nucleic acid sequences, SNP alleles for example, at asingle nucleotide mismatch (e.g., a mismatch between paralogoussequences, or SNP alleles). The terms “paralogous sequence” or“paralogous sequences” refer to sequences that have a commonevolutionary origin but which may be duplicated over time in the genomeof interest. Paralogous sequences may conserve gene structure (e.g.,number and relative position of introns and exons and preferablytranscript length), as well as sequence. Therefore, the methodsdescribed herein can be used to detect sequence mismatches inSNP-alleles or in evolutionarily conserved regions that differ by one ormore point mutations, insertions or deletions (both will hereinafter bereferred to as “mismatch site” or “sequence mismatch”).

The mismatch may be detected by the incorporation of one or moredeoxynucleotides and/or dideoxynucleotides to a primer extension primeror oligonucleotide species, which hybridizes to a region adjacent to theSNP site (e.g., mismatch site). The extension oligonucleotide generallyis extended with a polymerase. In some embodiments, a detectable tag,detectable moiety or detectable moiety is incorporated into theextension oligonucleotide or into the nucleotides added on to theextension oligonucleotide (e.g., biotin or streptavidin). The extendedoligonucleotide can be detected by any known suitable detection process(e.g., mass spectrometry; sequencing processes). In some embodiments,the mismatch site is extended only by one or two complementarydeoxynucleotides or dideoxynucleotides that are tagged by a specificlabel or generate a primer extension product with a specific mass, andthe mismatch can be discriminated and quantified.

For embodiments using primer extension methods to amplify a targetsequence, the extension of the oligonucleotide species is not limited toa single round of extension, and is therefore distinguished from“one-time primer extension” described above. Non-limiting examples ofprimer extension or oligonucleotide extension methods suitable for usewith embodiments described herein are described in U.S. Pat. Nos.4,656,127; 4,851,331; 5,679,524; 5,834,189; 5,876,934; 5,908,755;5,912,118; 5,976,802; 5,981,186; 6,004,744; 6,013,431; 6,017,702;6,046,005; 6,087,095; 6,210,891; and WO 01/20039, for example.

A generalized description of an amplification process is presentedherein. Oligonucleotide species compositions described herein and targetnucleic acid are contacted, and complementary sequences anneal to oneanother, for example. Oligonucleotides can anneal to a nucleic acid, ator near (e.g., adjacent to, abutting, and the like) a target sequence ofinterest. A reaction mixture, containing all components necessary forfull enzymatic functionality, is added to the oligonucleotidespecies—target nucleic acid hybrid, and amplification can occur undersuitable conditions. Components of an amplification reaction mayinclude, but are not limited to, e.g., oligonucleotide speciescompositions (e.g., individual oligonucleotides, oligonucleotide pairs,oligonucleotide sets and the like) a polynucleotide template (e.g.,nucleic acid containing a target sequence), polymerase, nucleotides,dNTPs, an appropriate endonuclease and the like. Extension conditionsare sometimes a subset of, or substantially similar to amplificationconditions.

In some embodiments, non-naturally occurring nucleotides or nucleotideanalogs, such as analogs containing a detectable moiety or feature(e.g., fluorescent or colorimetric label) may be used, for example.Polymerases can be selected by a person of ordinary skill and includepolymerases for thermocycle amplification (e.g., Taq DNA Polymerase;Q-Bio™ Taq DNA Polymerase (recombinant truncated form of Taq DNAPolymerase lacking 5′-3′exo activity); SurePrime™ Polymerase (chemicallymodified Taq DNA polymerase for “hot start” PCR); Arrow™ Taq DNAPolymerase (high sensitivity and long template amplification)) andpolymerases for thermostable amplification (e.g., RNA polymerase fortranscription-mediated amplification (TMA) described at World Wide WebURL “gen-probe.com/pdfs/tma_whiteppr.pdf”). Other enzyme components canbe added, such as reverse transcriptase for transcription mediatedamplification (TMA) reactions, for example.

The terms “near” or “adjacent to” when referring to a nucleotide targetsequence refers to a distance or region between the end of the primerand the nucleotide or nucleotides of interest. As used herein adjacentis in the range of about 5 nucleotides to about 500 nucleotides (e.g.,about 5 nucleotides away from nucleotide of interest, about 10, about20, about 30, about 40, about 50, about 60, about 70, about 80, about90, about 100, about 150, about 200, about 250, about 300, abut 350,about 400, about 450 or about 500 nucleotides from a nucleotide ofinterest).

Each amplified nucleic acid species independently can be about 10 toabout 1000 base pairs in length in some embodiments. In certainembodiments, an amplified nucleic acid species is about 20 to about 250base pairs in length, sometimes is about 50 to about 150 base pairs inlength and sometimes is about 100 base pairs in length. Thus, in someembodiments, the length of each of the amplified nucleic acid speciesproducts independently is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104,106, 108, 110, 112, 114, 116, 118, 120, 125, 130, 135, 140, 145, 150,175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800,850, 900, 950 or 1000 base pairs (bp) in length.

An amplification product may include naturally occurring nucleotides,non-naturally occurring nucleotides, nucleotide analogs and the like andcombinations of the foregoing. An amplification product often has anucleotide sequence that is identical to or substantially identical to atarget sequence or complement thereof. A “substantially identical”nucleotide sequence in an amplification product will generally have ahigh degree of sequence identity to the nucleotide sequence speciesbeing amplified or complement thereof (e.g., about 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or greater than 99% sequenceidentity), and variations sometimes are a result of infidelity of thepolymerase used for extension and/or amplification, or additionalnucleotide sequence(s) added to the primers used for amplification.

PCR conditions can be dependent upon primer sequences, target abundance,and the desired amount of amplification, and therefore, one of skill inthe art may choose from a number of PCR protocols available (see, e.g.,U.S. Pat. Nos. 4,683,195 and 4,683,202; and PCR Protocols: A Guide toMethods and Applications, Innis et al., eds, 1990. PCR often is carriedout as an automated process with a thermostable enzyme. In this process,the temperature of the reaction mixture is cycled through a denaturingregion, a primer-annealing region, and an extension reaction regionautomatically. Machines specifically adapted for this purpose arecommercially available. A non-limiting example of a PCR protocol thatmay be suitable for embodiments described herein is, treating the sampleat 95° C. for 5 minutes; repeating forty-five cycles of 95° C. for 1minute, 59° C. for 1 minute, 10 seconds, and 72° C. for 1 minute 30seconds; and then treating the sample at 72° C. for 5 minutes.Additional PCR protocols are described in the example section.

Multiple cycles frequently are performed using a commercially availablethermal cycler. Suitable isothermal amplification processes known andselected by the person of ordinary skill in the art also may be applied,in certain embodiments.

In some embodiments, multiplex amplification processes may be used toamplify target sequences, such that multiple amplicons aresimultaneously amplified in a single, homogenous reaction. As usedherein “multiplex amplification” refers to a variant of PCR wheresimultaneous amplification of many target sequences in one reactionvessel may be accomplished by using more than one pair of primers (e.g.,more than one primer set). Multiplex amplification may be useful foranalysis of deletions, mutations, and polymorphisms, or quantitativeassays, in some embodiments. In certain embodiments multiplexamplification may be used for detecting paralog sequence imbalance,genotyping applications where simultaneous analysis of multiple markersis required, detection of pathogens or genetically modified organisms,or for microsatellite analyses. In some embodiments multiplexamplification may be combined with another amplification (e.g., PCR)method (e.g., nested PCR or hot start PCR, for example) to increaseamplification specificity and reproducibility. In some embodiments,multiplex amplification processes may be used to amplify theY-chromosome loci described herein.

In certain embodiments, nucleic acid amplification can generateadditional nucleic acid species of different or substantially similarnucleic acid sequence. In certain embodiments described herein,contaminating or additional nucleic acid species, which may containsequences substantially complementary to, or may be substantiallyidentical to, the target sequence, can be useful for sequencequantification, with the proviso that the level of contaminating oradditional sequences remains constant and therefore can be a reliablemarker whose level can be substantially reproduced. Additionalconsiderations that may affect sequence amplification reproducibilityare; PCR conditions (number of cycles, volume of reactions, meltingtemperature difference between primers pairs, and the like),concentration of target nucleic acid in sample (e.g. fetal nucleic acidin maternal nucleic acid background, viral nucleic acid in hostbackground), the number of chromosomes on which the nucleotide speciesof interest resides (e.g., paralogous sequences or SNP-alleles),variations in quality of prepared sample, and the like. The terms“substantially reproduced” or “substantially reproducible” as usedherein refer to a result (e.g., quantifiable amount of nucleic acid)that under substantially similar conditions would occur in substantiallythe same way about 75% of the time or greater, about 80%, about 85%,about 90%, about 95%, or about 99% of the time or greater.

In some embodiments, amplification may be performed on a solid support.In some embodiments, primers may be associated with a solid support. Incertain embodiments, target nucleic acid (e.g., template nucleic acid ortarget sequences) may be associated with a solid support. A nucleic acid(primer or target) in association with a solid support often is referredto as a solid phase nucleic acid.

In some embodiments, nucleic acid molecules provided for amplificationare in a “microreactor”. As used herein, the term “microreactor” refersto a partitioned space in which a nucleic acid molecule can hybridize toa solid support nucleic acid molecule. Examples of microreactorsinclude, without limitation, an emulsion globule (described hereafter)and a void in a substrate. A void in a substrate can be a pit, a pore ora well (e.g., microwell, nanowell, picowell, micropore, or nanopore) ina substrate constructed from a solid material useful for containingfluids (e.g., plastic (e.g., polypropylene, polyethylene, polystyrene)or silicon) in certain embodiments. Emulsion globules are partitioned byan immiscible phase as described in greater detail hereafter. In someembodiments, the microreactor volume is large enough to accommodate onesolid support (e.g., bead) in the microreactor and small enough toexclude the presence of two or more solid supports in the microreactor.

The term “emulsion” as used herein refers to a mixture of two immiscibleand unblendable substances, in which one substance (the dispersed phase)often is dispersed in the other substance (the continuous phase). Thedispersed phase can be an aqueous solution (i.e., a solution comprisingwater) in certain embodiments. In some embodiments, the dispersed phaseis composed predominantly of water (e.g., greater than 70%, greater than75%, greater than 80%, greater than 85%, greater than 90%, greater than95%, greater than 97%, greater than 98% and greater than 99% water (byweight)). Each discrete portion of a dispersed phase, such as an aqueousdispersed phase, is referred to herein as a “globule” or “microreactor.”A globule sometimes may be spheroidal, substantially spheroidal orsemi-spheroidal in shape, in certain embodiments.

The terms “emulsion apparatus” and “emulsion component(s)” as usedherein refer to apparatus and components that can be used to prepare anemulsion. Non-limiting examples of emulsion apparatus include withoutlimitation counter-flow, cross-current, rotating drum and membraneapparatus suitable for use by a person of ordinary skill to prepare anemulsion. An emulsion component forms the continuous phase of anemulsion in certain embodiments, and includes without limitation asubstance immiscible with water, such as a component comprising orconsisting essentially of an oil (e.g., a heat-stable, biocompatible oil(e.g., light mineral oil)). A biocompatible emulsion stabilizer can beutilized as an emulsion component. Emulsion stabilizers include withoutlimitation Atlox 4912, Span 80 and other biocompatible surfactants.

In some embodiments, components useful for biological reactions can beincluded in the dispersed phase. Globules of the emulsion can include(i) a solid support unit (e.g., one bead or one particle); (ii) samplenucleic acid molecule; and (iii) a sufficient amount of extension agentsto elongate solid phase nucleic acid and amplify the elongated solidphase nucleic acid (e.g., extension nucleotides, polymerase, primer). Insome embodiments, endonucleases and components necessary forendonuclease function may be included in the components useful forbiological reactions as described below in the example section. Inactiveglobules in the emulsion may include a subset of these components (e.g.,solid support and extension reagents and no sample nucleic acid) andsome can be empty (i.e., some globules will include no solid support, nosample nucleic acid and no extension agents).

Emulsions may be prepared using known suitable methods (e.g., Nakano etal. “Single-molecule PCR using water-in-oil emulsion;” Journal ofBiotechnology 102 (2003) 117-124). Emulsification methods includewithout limitation adjuvant methods, counter-flow methods, cross-currentmethods, rotating drum methods, membrane methods, and the like. Incertain embodiments, an aqueous reaction mixture containing a solidsupport (hereafter the “reaction mixture”) is prepared and then added toa biocompatible oil. In certain embodiments, the reaction mixture may beadded dropwise into a spinning mixture of biocompatible oil (e.g., lightmineral oil (Sigma)) and allowed to emulsify. In some embodiments, thereaction mixture may be added dropwise into a cross-flow ofbiocompatible oil. The size of aqueous globules in the emulsion can beadjusted, such as by varying the flow rate and speed at which thecomponents are added to one another, for example.

The size of emulsion globules can be selected by the person of ordinaryskill in certain embodiments based on two competing factors: (i)globules are sufficiently large to encompass one solid support molecule,one sample nucleic acid molecule, and sufficient extension agents forthe degree of elongation and amplification required; and (ii) globulesare sufficiently small so that a population of globules can be amplifiedby conventional laboratory equipment (e.g., thermocycling equipment,test tubes, incubators and the like). Globules in the emulsion can havea nominal, mean or average diameter of about 5 microns to about 500microns, about 10 microns to about 350 microns, about 50 to 250 microns,about 100 microns to about 200 microns, or about 5, 10, 15, 20, 25, 30,35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400or 500 microns in certain embodiments.

In certain embodiments, amplified nucleic acid species in a set are ofidentical length, and sometimes the amplified nucleic acid species in aset are of a different length. For example, one amplified nucleic acidspecies may be longer than one or more other amplified nucleic acidspecies in the set by about 1 to about 100 nucleotides (e.g., about 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40,50, 60, 70, 80 or 90 nucleotides longer).

In some embodiments, a ratio can be determined for the amount of oneamplified nucleic acid species in a set to the amount of anotheramplified nucleic acid species in the set (hereafter a “set ratio”). Insome embodiments, the amount of one amplified nucleic acid species in aset is about equal to the amount of another amplified nucleic acidspecies in the set (i.e., amounts of amplified nucleic acid species in aset are about 1:1), which generally is the case when the number ofchromosomes or the amount of DNA representative of nucleic acid speciesin a sample bearing each nucleotide sequence species amplified is aboutequal. The term “amount” as used herein with respect to amplifiednucleic acid species refers to any suitable measurement, including, butnot limited to, copy number, weight (e.g., grams) and concentration(e.g., grams per unit volume (e.g., milliliter); molar units). In someembodiments, the ratio of fetal nucleic acid to maternal nucleic acid(or conversely maternal nucleic acid to fetal nucleic acid) can be usedin conjunction with measurements of the ratios of mismatch sequences fordetermination of chromosomal abnormalities possibly associated with sexchromosomes. That is, the percentage of fetal nucleic acid detected in amaternal nucleic acid background or the ratio of fetal to maternalnucleic acid in a sample, can be used to detect chromosomalaneuploidies.

In certain embodiments, the amount of one amplified nucleic acid speciesin a set can differ from the amount of another amplified nucleic acidspecies in a set, even when the number of chromosomes in a samplebearing each nucleotide sequence species amplified is about equal. Insome embodiments, amounts of amplified nucleic acid species within a setmay vary up to a threshold level at which a chromosome abnormality canbe detected with a confidence level of about 95% (e.g., about 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or greater than 99%). In certainembodiments, the amounts of the amplified nucleic acid species in a setvary by about 50% or less (e.g., about 45, 40, 35, 30, 25, 20, 15, 10,5, 4, 3, 2 or 1%, or less than 1%). Thus, in certain embodiments amountsof amplified nucleic acid species in a set may vary from about 1:1 toabout 1:1.5. Without being limited by theory, certain factors can leadto the observation that the amount of one amplified nucleic acid speciesin a set can differ from the amount of another amplified nucleic acidspecies in a set, even when the number of chromosomes in a samplebearing each nucleotide sequence species amplified is about equal. Suchfactors may include different amplification efficiency rates and/oramplification from a chromosome not intended in the assay design.

Each amplified nucleic acid species in a set generally is amplifiedunder conditions that amplify that species at a substantiallyreproducible level. The term “substantially reproducible level” as usedherein refers to consistency of amplification levels for a particularamplified nucleic acid species per unit template nucleic acid (e.g., perunit template nucleic acid that contains the particular nucleotidesequence species amplified). A substantially reproducible level variesby about 1% or less in certain embodiments, after factoring the amountof template nucleic acid giving rise to a particular amplificationnucleic acid species (e.g., normalized for the amount of templatenucleic acid). In some embodiments, a substantially reproducible levelvaries by 5%, 4%, 3%, 2%, 1.5%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, 0.005% or0.001% after factoring the amount of template nucleic acid giving riseto a particular amplification nucleic acid species.

In some embodiments amplification nucleic acid species (e.g., amplifiedtarget sequences) of oligonucleotide species composition sets describedherein may be generated in one reaction vessel. In some embodimentsamplification of mismatch sequences may be performed in a singlereaction vessel. In certain embodiments, mismatch sequences (on the sameor different chromosomes) may be amplified by a single oligonucleotidespecies pair or set. In some embodiments target sequences may beamplified by a single oligonucleotide species pair or set.

In some embodiments target sequences in a set may be amplified with twoor more oligonucleotide species pairs. In some embodiments a subsequenceof a target nucleic acid may be amplified using a single oligonucleotidespecies pair or set. In some embodiments a subsequence of a targetnucleic acid may be amplified using two or more oligonucleotide speciespairs.

Polymerase Extendable Oligonucleotide Species Compositions

In certain embodiments, relatively short nucleic acid of a nucleic acidcomposition is enriched using methods and compositions described herein,and the all, or a subset of, the enriched relatively short nucleic acidis analyzed. An oligonucleotide species that hybridizes to one or morenucleic acids (e.g., target nucleic acids) in the enriched nucleic acidsometimes are utilized. Oligonucleotide species can be useful foramplification, detection, quantification and sequencing of targetnucleic acids. In some embodiments the oligonucleotide speciescompositions may be complementary to, and hybridize or annealspecifically to or near (e.g., adjacent to) sequences that flank atarget region therein. In some embodiments the oligonucleotide speciescompositions described herein are used in sets, where a set contains atleast a pair. In some embodiments a set of oligonucleotide species mayinclude a third or a fourth nucleic acid (e.g., two pairs ofoligonucleotide species or nested sets of oligonucleotide species, forexample). A plurality of oligonucleotide species pairs may constitute aprimer set in certain embodiments (e.g., about 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95or 100 pairs). In some embodiments a plurality of oligonucleotidespecies sets, each set comprising pair(s) of primers, may be used.

The term “oligonucleotide species” as used herein refers to a nucleicacid that comprises a nucleotide sequence capable of hybridizing orannealing to a target nucleic acid, at or near (e.g., adjacent to) aspecific region of interest. As used herein, the term “PCRoligonucleotide species composition(s)” refers to oligonucleotides thatcan be used in a polymerase chain reaction (PCR) to amplify a targetnucleotide sequence, for example. In certain embodiments, at least oneof the PCR oligonucleotide species for amplification of a nucleotidesequence encoding a target nucleic acid can be a sequence-specificoligonucleotide species composition. In some embodiments,oligonucleotide species compositions described herein may be modified(e.g., addition of a universal primer sequence) to improve multiplexing.

Oligonucleotide species compositions described herein can allow forspecific determination of a target nucleic acid nucleotide sequence ordetection of the target nucleic acid sequence (e.g., presence or absenceof a sequence or copy number of a sequence), or feature thereof, forexample. Oligonucleotide species compositions described herein may alsobe used to detect amplification products or extension products, incertain embodiments. The oligonucleotide compositions and methods of usedescribed herein are useful for minimizing or eliminating extensionand/or amplification artifacts (e.g., “primer-dimers” and artifactscaused by annealing and extension during temperature transitions in aPCR thermocycling profile, for example) that can sometimes occur innucleic acid extension or amplification based assays. Theoligonucleotide species compositions described herein includeendonuclease cleavage sites for thermostable endonucleases that can beused in methods (single tube assays, multiplexed assays and the like),also described herein, that combine hybridization, cleavage andextension or amplification conditions to allow specific targetidentification and/or amplification.

The oligonucleotide species compositions described herein are oftensynthetic, but naturally occurring nucleic acid sequences with similarstructure and/or function may be used, in some embodiments. The term“specific”, “specifically” or “specificity”, as used herein with respectto nucleic acids, refers to the binding or hybridization of one moleculeto another molecule, such as a primer for a target polynucleotidesequence. That is, “specific”, “specifically” or “specificity” refers tothe recognition, contact, and formation of a stable complex between twomolecules, as compared to substantially less recognition, contact, orcomplex formation of either of those two molecules with other molecules.As used herein, the term “anneal” refers to the formation of a stablecomplex between two molecules. The terms “oligonucleotide species”,“oligonucleotide species composition”, “oligonucleotide composition”,“primer”, “oligo”, or “oligonucleotide” may be used interchangeablythroughout the document, when referring to primers.

The oligonucleotide species compositions described herein can bedesigned and synthesized using suitable processes, and may be of anylength suitable for hybridizing to a nucleotide sequence of interest(e.g., where the nucleic acid is in liquid phase or bound to a solidsupport) and performing analysis processes described herein.Oligonucleotide species compositions described herein may be designedbased upon a target nucleotide sequence. An oligonucleotide speciescomposition in some embodiments may be about 10 to about 100nucleotides, about 10 to about 70 nucleotides, about 10 to about 50nucleotides, about 15 to about 30 nucleotides, or about 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95 or 100 nucleotides in length. Anoligonucleotide species composition may be composed of naturallyoccurring and/or non-naturally occurring nucleotides (e.g., labelednucleotides), or a mixture thereof. Oligonucleotide species compositionembodiments suitable for use with method embodiments described hereinmay be synthesized and labeled using known techniques. Oligonucleotides(e.g., primers) may be chemically synthesized according to the solidphase phosphoramidite triester method first described by Beaucage andCaruthers, Tetrahedron Letts., 22:1859-1862, 1981, using an automatedsynthesizer, as described in Needham-VanDevanter et al., Nucleic AcidsRes. 12:6159-6168, 1984. Purification of oligonucleotides can beeffected by native acrylamide gel electrophoresis or by anion-exchangehigh-performance liquid chromatography (HPLC), for example, as describedin Pearson and Regnier, J. Chrom., 255:137-149, 1983.

All or a portion of an oligonucleotide species composition nucleic acidsequence (naturally occurring or synthetic) may be substantiallycomplementary to a target nucleic acid sequence, in some embodiments. Asreferred to herein, “substantially complementary” with respect tosequences refers to nucleotide sequences that will hybridize with eachother. The stringency of the hybridization conditions can be altered totolerate varying amounts of sequence mismatch. Included are regions ofcounterpart, target and capture nucleotide sequences 55% or more, 56% ormore, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more,62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% ormore, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more,73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% ormore, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more,84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% ormore, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more,95% or more, 96% or more, 97% or more, 98% or more or 99% or morecomplementary to each other.

Oligonucleotide compositions that contain subsequences that aresubstantially complimentary to a target nucleic acid sequence are alsosubstantially identical to the compliment of the target nucleic acidsequence. That is, primers can be substantially identical to theanti-sense strand of the nucleic acid. As referred to herein,“substantially identical” with respect to sequences refers to nucleotidesequences that are 55% or more, 56% or more, 57% or more, 58% or more,59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% ormore, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more,70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% ormore, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more,81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% ormore, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more,92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% ormore, 98% or more or 99% or more identical to each other. One test fordetermining whether two nucleotide sequences are substantially identicalis to determine the percent of identical nucleotide sequences shared.

Oligonucleotide species sequences and length may affect hybridization totarget nucleic acid sequences. Depending on the degree of mismatchbetween the oligonucleotide species and target nucleic acid, low, mediumor high stringency conditions may be used to effectoligonucleotide/target annealing. As used herein, the term “stringentconditions” refers to conditions for hybridization and washing. Methodsfor hybridization reaction temperature condition optimization are knownto those of skill in the art, and may be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y., 6.3.1-6.3.6 (1989). Aqueousand non-aqueous methods are described in that reference and either canbe used. Non-limiting examples of stringent hybridization conditions arehybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50° C.Another example of stringent hybridization conditions are hybridizationin 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed byone or more washes in 0.2×SSC, 0.1% SDS at 55° C. A further example ofstringent hybridization conditions is hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2×SSC, 0.1% SDS at 60° C. Often, stringent hybridizationconditions are hybridization in 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at65° C. More often, stringency conditions are 0.5M sodium phosphate, 7%SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65°C. Stringent hybridization temperatures can also be altered (i.e.lowered) with the addition of certain organic solvents, formamide forexample. Organic solvents, like formamide, reduce the thermal stabilityof double-stranded polynucleotides, so that hybridization can beperformed at lower temperatures, while still maintaining stringentconditions and extending the useful life of nucleic acids that may beheat labile.

In embodiments using extension or amplification methods describedherein, “stringent conditions” can also refer to conditions under whichan intact oligonucleotide species composition can anneal to a targetnucleic acid, but where one or more cleaved fragments of theoligonucleotide species composition cannot anneal to the target nucleicacid (e.g., intact oligonucleotide anneals at 65 C and one or morefragments anneals at 50 C). In some embodiments, the “stringentconditions” for extension and/or amplification methods described hereinare; substantially similar to, a subset of, or include as a subset,hybridization conditions, cleavage conditions, extension conditions,amplification conditions or combinations thereof.

As used herein, the phrase “hybridizing” or grammatical variationsthereof, refers to binding of a first nucleic acid molecule to a secondnucleic acid molecule under low, medium or high stringency conditions,or under nucleic acid synthesis conditions. Hybridizing can includeinstances where a first nucleic acid molecule binds to a second nucleicacid molecule, where the first and second nucleic acid molecules arecomplementary. As used herein, “specifically hybridizes” refers topreferential hybridization under nucleic acid synthesis conditions of anoligonucleotide species, to a nucleic acid molecule having a sequencecomplementary to the oligonucleotide species compared to hybridizationto a nucleic acid molecule not having a complementary sequence. Forexample, specific hybridization includes the hybridization of anoligonucleotide species composition to a target nucleic acid sequencethat is complementary to at least a portion of the oligonucleotidespecies composition.

In some embodiments oligonucleotide species compositions can include anucleotide subsequence that may be complementary to a solid phasenucleic acid oligonucleotide hybridization sequence or substantiallycomplementary to a solid phase nucleic acid primer hybridizationsequence (e.g., about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or greater than 99% identical to the primer hybridization sequencecomplement when aligned). An oligonucleotide species composition maycontain a nucleotide subsequence not complementary to or notsubstantially complementary to a solid phase nucleic acidoligonucleotide hybridization sequence (e.g., at the 3′ or 5′ end of thenucleotide subsequence in the oligonucleotide species compositioncomplementary to or substantially complementary to the solid phaseoligonucleotide hybridization sequence).

An oligonucleotide species composition, in certain embodiments, maycontain a detectable feature, moiety, molecule or entity (e.g., afluorophore, radioisotope, colorimetric agent, particle, enzyme and thelike). In some embodiments, a detectable feature may be a capture agentor a blocking agent. In some embodiments each oligonucleotide speciesmay contain a blocking moiety. In some embodiments the blocking moietyof a first oligonucleotide species is different than the blocking moietyof a second oligonucleotide species. Non-limiting examples of blockingagents include; phosphate group, thiol group, phosphorothioate group,amino modifier, biotin, biotin-TEG, cholesteryl-TEG, digoxigenin NHSester, thiol modifier C3 S—S(Disulfide), inverted dT, C3 spacer and thelike. In some embodiments more than one blocking group can beincorporated into an oligonucleotide species at, or near, one moreendonuclease cleavage sites to allow the oligonucleotide speciescomposition to be sequentially deblocked to allow multiple rounds ofextension.

When desired, the nucleic acid can be modified to include a detectablefeature or blocking moiety using any method known to one of skill in theart. The feature may be incorporated as part of the synthesis, or addedon prior to using the oligonucleotide species composition in any of theprocesses described herein. Incorporation of a detectable feature may beperformed either in liquid phase or on solid phase. In some embodimentsthe detectable feature may be useful for detection of targets. In someembodiments the detectable feature may be useful for the quantificationtarget nucleic acids (e.g., determining copy number of a particularsequence or species of nucleic acid). Any detectable feature suitablefor detection of an interaction or biological activity in a system canbe appropriately selected and utilized by the artisan. Examples ofdetectable features are fluorescent labels such as fluorescein,rhodamine, and others (e.g., Anantha, et al., Biochemistry (1998)37:2709 2714; and Qu & Chaires, Methods Enzymol. (2000) 321:353 369);radioactive isotopes (e.g., 125I, 131I, 35S, 31P, 32P, 33P, 14C, 3H,7Be, 28Mg, 57Co, 65Zn, 67Cu, 68Ge, 82Sr, 83Rb, 95Tc, 96Tc, 103Pd, 109Cd,and 127Xe); light scattering labels (e.g., U.S. Pat. No. 6,214,560, andcommercially available from Genicon Sciences Corporation, CA);chemiluminescent labels and enzyme substrates (e.g., dioxetanes andacridinium esters), enzymic or protein labels (e.g., green fluorescenceprotein (GFP) or color variant thereof, luciferase, peroxidase); otherchromogenic labels or dyes (e.g., cyanine), and other cofactors orbiomolecules such as digoxigenin, strepdavidin, biotin (e.g., members ofa binding pair such as biotin and avidin for example), affinity capturemoieties, 3′ blocking agents (e.g., phosphate group, thiol group,phosphorothioate, amino modifier, biotin, biotin-TEG, cholesteryl-TEG,digoxigenin NHS ester, thiol modifier C3 S—S(Disulfide), inverted dT, C3spacer) and the like. In some embodiments an oligonucleotide speciescomposition may be labeled with an affinity capture moiety. Alsoincluded in detectable features are those labels useful for massmodification for detection with mass spectrometry (e.g., matrix-assistedlaser desorption ionization (MALDI) mass spectrometry and electrospray(ES) mass spectrometry).

An oligonucleotide species composition also may refer to apolynucleotide sequence that hybridizes to a subsequence of a targetnucleic acid or another oligonucleotide species and facilitates thedetection of an oligonucleotide, a target nucleic acid or both, andamplification products or extension products, as with molecular beacons,for example. The term “molecular beacon” as used herein refers todetectable molecule, wherein the detectable feature, or property, of themolecule is detectable only under certain specific conditions, therebyenabling it to function as a specific and informative signal.Non-limiting examples of detectable properties are optical properties,electrical properties, magnetic properties, chemical properties and timeor speed through an opening of known size.

In some embodiments a molecular beacon can be a single-strandedoligonucleotide capable of forming a stem-loop structure, where the loopsequence may be complementary to a target nucleic acid sequence ofinterest and is flanked by short complementary arms that can form astem. The oligonucleotide may be labeled at one end with a fluorophoreand at the other end with a quencher molecule. In the stem-loopconformation, energy from the excited fluorophore is transferred to thequencher, through long-range dipole-dipole coupling similar to that seenin fluorescence resonance energy transfer, or FRET, and released as heatinstead of light. When the loop sequence is hybridized to a specifictarget sequence, the two ends of the molecule are separated and theenergy from the excited fluorophore is emitted as light, generating adetectable signal. Molecular beacons offer the added advantage thatremoval of excess probe is unnecessary due to the self-quenching natureof the unhybridized probe. In some embodiments molecular beacon probescan be designed to either discriminate or tolerate mismatches betweenthe loop and target sequences by modulating the relative strengths ofthe loop-target hybridization and stem formation. As referred to herein,the term “mismatched nucleotide” or a “mismatch” refers to a nucleotidethat is not complementary to the target sequence at that position orpositions. A probe may have at least one mismatch, but can also have 2,3, 4, 5, 6 or 7 or more mismatched nucleotides.

In some embodiments the oligonucleotide species compositions describedherein can contain internal subsequences that may form stem-loopstructures, where the stem-loop sequences are not complementary to anysequence in the template DNA. The Tm of the internal structure is toolow for it to form a stem-loop structure, unless the two sides arebrought together by the annealing of the 5′ and 3′ ends to the template(e.g., the reverse of a molecular beacon).

Detection

Relatively short nucleic acid enriched by the methods and compositionsdescribed herein can be analyzed, in certain embodiments. For example,the presence, absence or amount of a particular nucleic acid (e.g.,target nucleic acid) or subsequence thereof (e.g., polymorphism) may bedetected in some embodiments. Thus, polymorphisms, polynucleotidesequences generated, amplified nucleic acid species (e.g. amplicons oramplification products) or detectable products (e.g., extensionproducts), prepared from the foregoing, can be detected by a suitabledetection process in some embodiments. Non-limiting examples of methodsof detection, quantification, sequencing and the like are; massdetection of mass modified amplicons (e.g., matrix-assisted laserdesorption ionization (MALDI) mass spectrometry and electrospray (ES)mass spectrometry), a primer extension method (e.g., iPLEX™; Sequenom,Inc.), microsequencing methods (e.g., a modification of primer extensionmethodology), ligase sequence determination methods (e.g., U.S. Pat.Nos. 5,679,524 and 5,952,174, and WO 01/27326), mismatch sequencedetermination methods (e.g., U.S. Pat. Nos. 5,851,770; 5,958,692;6,110,684; and 6,183,958), direct DNA sequencing, restriction fragmentlength polymorphism (RFLP analysis), allele specific oligonucleotide(ASO) analysis, methylation-specific PCR (MSPCR), pyrosequencinganalysis, acycloprime analysis, Reverse dot blot, GeneChip microarrays,Dynamic allele-specific hybridization (DASH), Peptide nucleic acid (PNA)and locked nucleic acids (LNA) probes, TaqMan, Molecular Beacons,Intercalating dye, FRET primers, AlphaScreen, SNPstream, genetic bitanalysis (GBA), Multiplex minisequencing, SNaPshot, GOOD assay,Microarray miniseq, arrayed primer extension (APEX), Microarray primerextension (e.g., microarray sequence determination methods), Tag arrays,Coded microspheres, Template-directed incorporation (TDI), fluorescencepolarization, Colorimetric oligonucleotide ligation assay (OLA),Sequence-coded OLA, Microarray ligation, Ligase chain reaction, Padlockprobes, Invader assay, hybridization methods (e.g., hybridization usingat least one probe, hybridization using at least one fluorescentlylabeled probe, and the like), conventional dot blot analyses, singlestrand conformational polymorphism analysis (SSCP, e.g., U.S. Pat. Nos.5,891,625 and 6,013,499; Orita et al., Proc. Natl. Acad. Sci. U.S.A 86:27776-2770 (1989)), denaturing gradient gel electrophoresis (DGGE),heteroduplex analysis, mismatch cleavage detection, and techniquesdescribed in Sheffield et al., Proc. Natl. Acad. Sci. USA 49: 699-706(1991), White et al., Genomics 12: 301-306 (1992), Grompe et al., Proc.Natl. Acad. Sci. USA 86: 5855-5892 (1989), and Grompe, Nature Genetics5: 111-117 (1993), cloning and sequencing, electrophoresis, the use ofhybridization probes and quantitative real time polymerase chainreaction (QRT-PCR), digital PCR, nanopore sequencing, chips andcombinations thereof. The detection and quantification of alleles orparalogs can be carried out using the “closed-tube” methods described inU.S. patent application Ser. No. 11/950,395, which was filed Dec. 4,2007. In some embodiments the amount of each amplified nucleic acidspecies is determined by mass spectrometry, primer extension, sequencing(e.g., any suitable method, for example nanopore or pyrosequencing),Quantitative PCR (Q-PCR or QRT-PCR), digital PCR, combinations thereof,and the like.

In addition to the methods of detection listed above, the followingdetection methods may also be used to detect amplified nucleic acidspecies (e.g., target sequences). In some embodiments, the amplifiednucleic acid species can be sequenced directly using any suitablenucleic acid sequencing method. Non-limiting examples of nucleic acidsequencing methods useful for process described herein are;pyrosequencing, nanopore based sequencing methods (e.g., sequencing bysynthesis), sequencing by ligation, sequencing by hybridization,microsequencing (primer extension based polymorphism detection), andconventional nucleotide sequencing (e.g., dideoxy sequencing usingconventional methods).

In some embodiments, the amplified sequence(s) may be cloned prior tosequence analysis. That is, the amplified nucleic acid species may beligated into a nucleic acid cloning vector by any process known to oneof skill in the art. Cloning of the amplified nucleic acid species maybe performed by including unique restriction sites in oligonucleotidespecies subsequences, which can be used to generate a fragment flankedby restriction sites useful for cloning into an appropriately preparedvector, in some embodiments. In certain embodiments blunt-ended cloningcan be used to clone amplified nucleic acid species into anappropriately prepared cloning vector. Cloning of the amplified nucleicacid species may be useful for further manipulation, modification,storage, and analysis of the target sequence of interest. In someembodiments, oligonucleotide species compositions may be designed tooverlap an SNP site to allow analysis by allele-specific PCR.

Allele-specific PCR may be used to discriminate between nucleic acids ina nucleic acid composition (e.g., fetal target in nucleic acid isolatedfrom maternal sample, for example), because only the correctlyhybridized primers will be amplified. In some embodiments, the amplifiednucleic acid species may be further analyzed by hybridization (e.g.,liquid or solid phase hybridization using sequence specific probes, forexample).

Amplified nucleic acids (including amplified nucleic acids that resultfrom reverse transcription) may be modified nucleic acids. Reversetranscribed nucleic acids also may be modified nucleic acids. Modifiednucleic acids can include nucleotide analogs, and in certain embodimentsinclude a detectable feature and/or a capture agent (e.g., biomoleculesor members of a binding pair, as listed below). In some embodiments thedetectable feature and the capture agent can be the same moiety.Modified nucleic acids can be detected by detecting a detectable featureor “signal-generating moiety” in some embodiments. The term“signal-generating” as used herein refers to any atom or molecule thatcan provide a detectable or quantifiable effect and that can be attachedto a nucleic acid. In certain embodiments, a detectable featuregenerates a unique light signal, a fluorescent signal, a luminescentsignal, an electrical property, a chemical property, a magnetic propertyand the like.

Detectable features include, but are not limited to, nucleotides(labeled or unlabelled), compomers, sugars, peptides, proteins,antibodies, chemical compounds, conducting polymers, binding moietiessuch as biotin, mass tags, colorimetric agents, light emitting agents,chemiluminescent agents, light scattering agents, fluorescent tags,radioactive tags, charge tags (electrical or magnetic charge), volatiletags and hydrophobic tags, biomolecules (e.g., members of a binding pairantibody/antigen, antibody/antibody, antibody/antibody fragment,antibody/antibody receptor, antibody/protein A or protein G,hapten/anti-hapten, biotin/avidin, biotin/streptavidin, folicacid/folate binding protein, vitamin B12/intrinsic factor, chemicalreactive group/complementary chemical reactive group (e.g.,sulfhydryl/maleimide, sulfhydryl/haloacetyl derivative,amine/isotriocyanate, amine/succinimidyl ester, and amine/sulfonylhalides) and the like, some of which are further described below. Insome embodiments a probe or oligonucleotide species may contain asignal-generating moiety that hybridizes to a target and alters thepassage of the target nucleic acid through a nanopore, and can generatea signal when released from the target nucleic acid when it passesthrough the nanopore (e.g., alters the speed or time through a pore ofknown size).

A solution containing amplicons produced by an amplification process, ora solution containing extension products produced by an extensionprocess, can be subjected to further processing. For example, a solutioncan be contacted with an agent that removes phosphate moieties from freenucleotides that have not been incorporated into an amplicon orextension product. An example of such an agent is a phosphatase (e.g.,alkaline phosphatase). Amplicons and extension products also may beassociated with a solid phase, may be washed, may be contacted with anagent that removes a terminal phosphate (e.g., exposure to aphosphatase), may be contacted with an agent that removes a terminalnucleotide (e.g., exonuclease), may be contacted with an agent thatcleaves (e.g., endonuclease, ribonuclease), and the like.

Mass spectrometry is a particularly effective method for the detectionof nucleic acids (e.g., PCR amplicon, primer extension product, detectorprobe cleaved from a target nucleic acid). Presence of a target nucleicacid is verified by comparing the mass of the detected signal with theexpected mass of the target nucleic acid. The relative signal strength,e.g., mass peak on a spectra, for a particular target nucleic acidindicates the relative population of the target nucleic acid amongstother nucleic acids, thus enabling calculation of a ratio of target toother nucleic acid or sequence copy number directly from the data. For areview of genotyping methods using Sequenom® standard iPLEX™ assay andMassARRAY® technology, see Jurinke, C., Oeth, P., van den Boom, D.,“MALDI-TOF mass spectrometry: a versatile tool for high-performance DNAanalysis.” Mol. Biotechnol. 26, 147-164 (2004); and Oeth, P. et al.,“iPLEX™ Assay: Increased Plexing Efficiency and Flexibility forMassARRAY® System through single base primer extension withmass-modified Terminators.” SEQUENOM Application Note (2005). For areview of detecting and quantifying target nucleic using cleavabledetector probes (e.g., oligonucleotide compositions described herein)that are cleaved during the amplification process and detected by massspectrometry, see U.S. patent application Ser. No. 11/950,395, which wasfiled Dec. 4, 2007, and is hereby incorporated by reference. Suchapproaches may be adapted to detection of chromosome abnormalities usingoligonucleotide species compositions and methods described herein.

In some embodiments, amplified nucleic acid species may be detected by(a) contacting the amplified nucleic acid species (e.g., amplicons) withextension oligonucleotide species compositions (e.g., detection ordetector oligonucleotides or primers), (b) preparing extended extensionoligonucleotide species compositions, and (c) determining the relativeamount of the one or more mismatch nucleotides (e.g., SNP that existbetween SNP-alleles or paralogous sequences) by analyzing the extendeddetection oligonucleotide species compositions (e.g., extensionoligonucleotides, or detection of extension products). In certainembodiments one or more mismatch nucleotides may be analyzed by massspectrometry. In some embodiments amplification, using methods describedherein, may generate between about 1 to about 100 amplicon sets, about 2to about 80 amplicon sets, about 4 to about 60 amplicon sets, about 6 toabout 40 amplicon sets, and about 8 to about 20 amplicon sets (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, or about 100 amplicon sets).

An example using mass spectrometry for detection of amplicon sets (e.g.,sets of amplification products) is presented herein. Amplicons may becontacted (in solution or on solid phase) with a set of oligonucleotides(the same oligonucleotide species compositions used for amplification ordifferent oligonucleotides representative of subsequences in the oligoor target nucleic acid) under hybridization conditions, where: (1) eacholigonucleotide in the set comprises a hybridization sequence capable ofspecifically hybridizing to one amplicon under the hybridizationconditions when the amplicon is present in the solution, (2) eacholigonucleotide in the set comprises a distinguishable tag located 5′ ofthe hybridization sequence, (3) a feature of the distinguishable tag ofone oligonucleotide detectably differs from the features ofdistinguishable tags of other oligonucleotides in the set; and (4) eachdistinguishable tag specifically corresponds to a specific amplicon andthereby specifically corresponds to a specific target nucleic acid. Thehybridized amplicon and “detection” oligonucleotide species aresubjected to nucleotide synthesis conditions that allow extension of thedetection oligonucleotide by one or more nucleotides (labeled with adetectable entity or moiety, or unlabeled), where one of the one or morenucleotides can be a terminating nucleotide. In some embodiments one ormore of the nucleotides added to the oligonucleotide species maycomprises a capture agent. In embodiments where hybridization occurredin solution, capture of the oligo/amplicon to solid support may bedesirable. The detectable moieties or entities can be released from theextended detection oligonucleotide species composition, and detection ofthe moiety determines the presence, absence, copy number of thenucleotide sequence of interest, or in some embodiments can provideinformation regarding the status of a reaction. In certain embodiments,the extension may be performed once yielding one extendedoligonucleotide. In some embodiments, the extension may be performedmultiple times (e.g., under amplification conditions) yielding multiplecopies of the extended oligonucleotide. In some embodiments performingthe extension multiple times can produce a sufficient number of copiessuch that interpretation of signals, representing copy number of aparticular sequence, can be made with a confidence level of 95% or more(e.g., confidence level of 95% or more, 96% or more, 97% or more, 98% ormore, 99% or more, or a confidence level of 99.5% or more). In someembodiments, the method for detecting amplicon sets can be used todetect extension products.

Methods provided herein allow for high-throughput detection of nucleicacid species in a plurality of nucleic acids (e.g., nucleotide sequencespecies, amplified nucleic acid species and detectable productsgenerated from the foregoing). Multiplexing refers to the simultaneousdetection of more than one nucleic acid species. General methods forperforming multiplexed reactions in conjunction with mass spectrometryare known (see, e.g., U.S. Pat. Nos. 6,043,031, 5,547,835 andInternational PCT application No. WO 97/37041). Multiplexing provides anadvantage that a plurality of nucleic acid species (e.g., some havingdifferent sequence variations) can be identified in as few as a singlemass spectrum, as compared to having to perform a separate massspectrometry analysis for each individual target nucleic acid species.Methods provided herein lend themselves to high-throughput, highlyautomated processes for analyzing sequence variations with high speedand accuracy, in some embodiments. In certain embodiments, methodsherein may be multiplexed at high levels in a single reaction.

Microarrays may be adapted for use with oligonucleotide speciescompositions and method embodiments described herein. A microarray canbe utilized for determining whether a polymorphic variant is present orabsent in a nucleic acid sample. A microarray may include anyoligonucleotides species compositions described herein, and methods formaking and using oligonucleotide microarrays suitable for prognostic useare disclosed in U.S. Pat. Nos. 5,492,806; 5,525,464; 5,589,330;5,695,940; 5,849,483; 6,018,041; 6,045,996; 6,136,541; 6,142,681;6,156,501; 6,197,506; 6,223,127; 6,225,625; 6,229,911; 6,239,273; WO00/52625; WO 01/25485; and WO 01/29259. The microarray typicallycomprises a solid support and the oligonucleotides may be linked to thissolid support by covalent bonds or by non-covalent interactions. Theoligonucleotides may also be linked to the solid support directly or bya spacer molecule. A microarray may comprise one or moreoligonucleotides complementary to a polymorphic target nucleic acidsite. Microarrays may be used with multiplexed protocols describedherein.

In certain embodiments, the number of nucleic acid species multiplexedinclude, without limitation, about 1 to about 500 (e.g., about 1-3, 3-5,5-7, 7-9, 9-11, 11-13, 13-15, 15-17, 17-19, 19-21, 21-23, 23-25, 25-27,27-29, 29-31, 31-33, 33-35, 35-37, 37-39, 39-41, 41-43, 43-45, 45-47,47-49, 49-51, 51-53, 53-55, 55-57, 57-59, 59-61, 61-63, 63-65, 65-67,67-69, 69-71, 71-73, 73-75, 75-77, 77-79, 79-81, 81-83, 83-85, 85-87,87-89, 89-91, 91-93, 93-95, 95-97, 97-101, 101-103, 103-105, 105-107,107-109, 109-111, 111-113, 113-115, 115-117, 117-119, 121-123, 123-125,125-127, 127-129, 129-131, 131-133, 133-135, 135-137, 137-139, 139-141,141-143, 143-145, 145-147, 147-149, 149-151, 151-153, 153-155, 155-157,157-159, 159-161, 161-163, 163-165, 165-167, 167-169, 169-171, 171-173,173-175, 175-177, 177-179, 179-181, 181-183, 183-185, 185-187, 187-189,189-191, 191-193, 193-195, 195-197, 197-199, 199-201, 201-203, 203-205,205-207, 207-209, 209-211, 211-213, 213-215, 215-217, 217-219, 219-221,221-223, 223-225, 225-227, 227-229, 229-231, 231-233, 233-235, 235-237,237-239, 239-241, 241-243, 243-245, 245-247, 247-249, 249-251, 251-253,253-255, 255-257, 257-259, 259-261, 261-263, 263-265, 265-267, 267-269,269-271, 271-273, 273-275, 275-277, 277-279, 279-281, 281-283, 283-285,285-287, 287-289, 289-291, 291-293, 293-295, 295-297, 297-299, 299-301,301-303, 303-305, 305-307, 307-309, 309-311, 311-313, 313-315, 315-317,317-319, 319-321, 321-323, 323-325, 325-327, 327-329, 329-331, 331-333,333-335, 335-337, 337-339, 339-341, 341-343, 343-345, 345-347, 347-349,349-351, 351-353, 353-355, 355-357, 357-359, 359-361, 361-363, 363-365,365-367, 367-369, 369-371, 371-373, 373-375, 375-377, 377-379, 379-381,381-383, 383-385, 385-387, 387-389, 389-391, 391-393, 393-395, 395-397,397-401, 401-403, 403-405, 405-407, 407-409, 409-411, 411-413, 413-415,415-417, 417-419, 419-421, 421-423, 423-425, 425-427, 427-429, 429-431,431-433, 433-435, 435-437, 437-439, 439-441, 441-443, 443-445, 445-447,447-449, 449-451, 451-453, 453-455, 455-457, 457-459, 459-461, 461-463,463-465, 465-467, 467-469, 469-471, 471-473, 473-475, 475-477, 477-479,479-481, 481-483, 483-485, 485-487, 487-489, 489-491, 491-493, 493-495,495-497, 497-501).

Design methods for achieving resolved mass spectra with multiplexedassays often include primer and oligonucleotide species compositiondesign methods and reaction design methods. For primer andoligonucleotide species composition design in multiplexed assays, thesame general guidelines for oligonucleotide species composition designapply for uniplexed reactions. The oligonucleotide species compositionsdescribed herein are designed to minimize or eliminate artifacts, thusavoiding false priming and primer dimers, the only difference being moreoligonucleotides species are involved for multiplex reactions. For massspectrometry applications, analyte peaks in the mass spectra for oneassay are sufficiently resolved from a product of any assay with whichthat assay is multiplexed, including pausing peaks and any otherby-product peaks. Also, analyte peaks optimally fall within auser-specified mass window, for example, within a range of 5,000-8,500Da. In some embodiments multiplex analysis may be adapted to massspectrometric detection of chromosome abnormalities, for example. Incertain embodiments multiplex analysis may be adapted to various singlenucleotide or nanopore based sequencing methods described herein.Commercially produced micro-reaction chambers or devices or arrays orchips may be used to facilitate multiplex analysis, and are commerciallyavailable.

Nucleotide sequence species, amplified nucleic acid species, ordetectable products generated from the foregoing may be subject tosequence analysis. The term “sequence analysis” as used herein refers todetermining a nucleotide sequence of an extension or amplificationproduct. The entire sequence or a partial sequence of an extension oramplification product can be determined, and the determined nucleotidesequence is referred to herein as a “read.” For example, one-time“primer extension” products or linear amplification products may beanalyzed directly without further amplification in some embodiments(e.g., by using single-molecule sequencing methodology (described ingreater detail hereafter)). In certain embodiments, linear amplificationproducts may be subject to further amplification and then analyzed(e.g., using sequencing by ligation or pyrosequencing methodology(described in greater detail hereafter)). Reads may be subject todifferent types of sequence analysis. Any suitable sequencing method canbe utilized to detect, and determine the amount of, nucleotide sequencespecies, amplified nucleic acid species, or detectable productsgenerated from the foregoing. Examples of certain sequencing methods aredescribed hereafter.

The terms “sequence analysis apparatus” and “sequence analysiscomponent(s)” used herein refer to apparatus, and one or more componentsused in conjunction with such apparatus, that can be used by a person ofordinary skill to determine a nucleotide sequence from amplificationproducts resulting from processes described herein (e.g., linear and/orexponential amplification products). Examples of sequencing platformsinclude, without limitation, the 454 platform (Roche) (Margulies, M. etal. 2005 Nature 437, 376-380), IIlumina Genomic Analyzer (or Solexaplatform) or SOLID System (Applied Bios stems) or the Helicos TrueSingle Molecule DNA sequencing technology (Harris T D et al. 2008Science, 320, 106-109), the single molecule, real-time (SMRT™)technology of Pacific Biosciences, and nanopore sequencing (Soni G V andMeller A. 2007 Clin Chem 53: 1996-2001). Such platforms allow sequencingof many nucleic acid molecules isolated from a specimen at high ordersof multiplexing in a parallel manner (Dear Brief Funct Genomic Proteomic2003; 1: 397-416). Each of these platforms allows sequencing of clonallyexpanded or non-amplified single molecules of nucleic acid fragments.Certain platforms involve, for example, (i) sequencing by ligation ofdye-modified probes (including cyclic ligation and cleavage), (ii)pyrosequencing, and (iii) single-molecule sequencing. Nucleotidesequence species, amplification nucleic acid species and detectableproducts generated there from can be considered a “study nucleic acid”for purposes of analyzing a nucleotide sequence by such sequenceanalysis platforms.

Sequencing by ligation is a nucleic acid sequencing method that relieson the sensitivity of DNA ligase to base-pairing mismatch. DNA ligasejoins together ends of DNA that are correctly base paired. Combining theability of DNA ligase to join together only correctly base paired DNAends, with mixed pools of fluorescently labeled oligonucleotides orprimers, enables sequence determination by fluorescence detection.Longer sequence reads may be obtained by including primers containingcleavable linkages that can be cleaved after label identification.Cleavage at the linker removes the label and regenerates the 5′phosphate on the end of the ligated oligonucleotide species, preparingthe oligonucleotide for another round of ligation. In some embodimentsoligonucleotide species compositions may be labeled with more than onefluorescent label (e.g., 1 fluorescent label, 2, 3, or 4 fluorescentlabels).

An example of a system that can be used by a person of ordinary skillbased on sequencing by ligation generally involves the following steps.Clonal bead populations can be prepared in emulsion microreactorscontaining target nucleic acid sequences (“template”), amplificationreaction components (e.g., including cleavage reaction components whereapplicable), beads and oligonucleotide species compositions describedherein. After amplification, templates are denatured and bead enrichmentis performed to separate beads with extended templates from undesiredbeads (e.g., beads with no extended templates). The template on theselected beads undergoes a 3′ modification to allow covalent bonding tothe slide, and modified beads can be deposited onto a glass slide.Deposition chambers offer the ability to segment a slide into one, fouror eight chambers during the bead loading process. For sequenceanalysis, primers hybridize to the adapter sequence. A set of four-colordye-labeled probes competes for ligation to the sequencingoligonucleotide species. Specificity of probe ligation is achieved byinterrogating every 4th and 5th base during the ligation series. Five toseven rounds of ligation, detection and cleavage record the color atevery 5th position with the number of rounds determined by the type oflibrary used. Following each round of ligation, a new complimentaryprimer offset by one base in the 5′ direction is laid down for anotherseries of ligations. Oligonucleotide species reset and ligation rounds(5-7 ligation cycles per round) are repeated sequentially five times togenerate 25-35 base pairs of sequence for a single tag. Wth mate-pairedsequencing, this process is repeated for a second tag. Such a system canbe used to exponentially amplify amplification products generated by aprocess described herein, e.g., by ligating a heterologous nucleic acidto the first amplification product generated by a process describedherein and performing emulsion amplification using the same or adifferent solid support originally used to generate the firstamplification product. Such a system also may be used to analyzeamplification products directly generated by a process described hereinby bypassing an exponential amplification process and directly sortingthe solid supports described herein on the glass slide.

Pyrosequencing is a nucleic acid sequencing method based on sequencingby synthesis, which relies on detection of a pyrophosphate released onnucleotide incorporation. Generally, sequencing by synthesis involvessynthesizing, one nucleotide at a time, a DNA strand complimentary tothe strand whose sequence is being sought. Target nucleic acids may beimmobilized to a solid support, hybridized with a sequencingoligonucleotide species (e.g., oligonucleotide species compositionsdescribed herein, for example), incubated with DNA polymerase, anappropriate endonuclease, ATP sulfurylase, luciferase, apyrase,adenosine 5′ phosphsulfate and luciferin. Nucleotide solutions aresequentially added and removed. Correct incorporation of a nucleotidereleases a pyrophosphate, which interacts with ATP sulfurylase andproduces ATP in the presence of adenosine 5′ phosphsulfate, fueling theluciferin reaction, which produces a chemiluminescent signal allowingsequence determination. The amount of light generated is proportional tothe number of bases added. Accordingly, the sequence downstream of thesequencing oligonucleotide species can be determined.

An example of a system that can be used by a person of ordinary skillbased on pyrosequencing generally involves the following steps: ligatingan adaptor nucleic acid to a study nucleic acid and hybridizing thestudy nucleic acid to a bead; amplifying a nucleotide sequence in thestudy nucleic acid in an emulsion; sorting beads using a picolitermultiwell solid support; and sequencing amplified nucleotide sequencesby pyrosequencing methodology (e.g., Nakano et al., “Single-molecule PCRusing water-in-oil emulsion;” Journal of Biotechnology 102: 117-124(2003)). Such a system can be used to exponentially amplifyamplification products generated by a process described herein, e.g., byligating a heterologous nucleic acid to the first amplification productgenerated by a process described herein.

Certain single-molecule sequencing embodiments are based on theprincipal of sequencing by synthesis, and utilize single-pairFluorescence Resonance Energy Transfer (single pair FRET) as a mechanismby which photons are emitted as a result of successful nucleotideincorporation. The emitted photons often are detected using intensifiedor high sensitivity cooled charge-couple-devices in conjunction withtotal internal reflection microscopy (TIRM). Photons are only emittedwhen the introduced reaction solution contains the correct nucleotidefor incorporation into the growing nucleic acid chain that issynthesized as a result of the sequencing process. In FRET basedsingle-molecule sequencing, energy is transferred between twofluorescent dyes, sometimes polymethine cyanine dyes Cy3 and Cy5,through long-range dipole interactions. The donor is excited at itsspecific excitation wavelength and the excited state energy istransferred, non-radiatively to the acceptor dye, which in turn becomesexcited. The acceptor dye eventually returns to the ground state byradiative emission of a photon. The two dyes used in the energy transferprocess represent the “single pair”, in single pair FRET. Cy3 often isused as the donor fluorophore and often is incorporated as the firstlabeled nucleotide. Cy5 often is used as the acceptor fluorophore and isused as the nucleotide label for successive nucleotide additions afterincorporation of a first Cy3 labeled nucleotide. The fluorophoresgenerally are within 10 nanometers of each for energy transfer to occursuccessfully.

An example of a system that can be used based on single-moleculesequencing generally involves hybridizing an oligonucleotide species toa target nucleic acid sequence to generate a complex; associating thecomplex with a solid phase; iteratively extending the oligonucleotidespecies by a nucleotide tagged with a fluorescent molecule; andcapturing an image of fluorescence resonance energy transfer signalsafter each iteration (e.g., U.S. Pat. No. 7,169,314; Braslaysky et al.,PNAS 100(7): 3960-3964 (2003)). Such a system can be used to directlysequence amplification products (linearly or exponentially amplifiedproducts) generated by processes described herein. In some embodimentsthe amplification products can be hybridized to an oligonucleotide thatcontains sequences complementary to immobilized capture sequencespresent on a solid support, a bead or glass slide for example.Hybridization of the oligonucleotide species-amplification productcomplexes with the immobilized capture sequences, immobilizesamplification products to solid supports for single pair FRET basedsequencing by synthesis. The oligonucleotide species often isfluorescent, so that an initial reference image of the surface of theslide with immobilized nucleic acids can be generated. The initialreference image is useful for determining locations at which truenucleotide incorporation is occurring. Fluorescence signals detected inarray locations not initially identified in the “primer only” referenceimage are discarded as non-specific fluorescence. Followingimmobilization of the oligonucleotide species-amplification productcomplexes, the bound nucleic acids often are sequenced in parallel bythe iterative steps of, a) polymerase extension in the presence of onefluorescently labeled nucleotide, b) detection of fluorescence usingappropriate microscopy, TIRM for example, c) removal of fluorescentnucleotide, and d) return to step a with a different fluorescentlylabeled nucleotide.

In some embodiments, nucleotide sequencing may be by solid phase singlenucleotide sequencing methods and processes. Solid phase singlenucleotide sequencing methods involve contacting target nucleic acid andsolid support under conditions in which a single molecule of samplenucleic acid hybridizes to a single molecule of a solid support. Suchconditions can include providing the solid support molecules and asingle molecule of target nucleic acid in a “microreactor.” Suchconditions also can include providing a mixture in which the targetnucleic acid molecule can hybridize to solid phase nucleic acid on thesolid support. Single nucleotide sequencing methods useful in theembodiments described herein are described in U.S. Provisional PatentApplication Ser. No. 61/021,871 filed Jan. 17, 2008.

In certain embodiments, nanopore sequencing detection methods include(a) contacting a target nucleic acid for sequencing (“base nucleicacid,” e.g., linked probe molecule) with sequence-specific detectors(e.g., oligonucleotide species compositions described herein), underconditions in which the detectors specifically hybridize tosubstantially complementary subsequences of the base nucleic acid; (b)detecting signals from the detectors and (c) determining the sequence ofthe base nucleic acid according to the signals detected. In certainembodiments, the detectors hybridized to the base nucleic acid aredisassociated from the base nucleic acid (e.g., sequentiallydissociated) when the detectors interfere with a nanopore structure asthe base nucleic acid passes through a pore, and the detectorsdisassociated from the base sequence are detected. In some embodiments,a detector disassociated from a base nucleic acid emits a detectablesignal, and the detector hybridized to the base nucleic acid emits adifferent detectable signal or no detectable signal. In certainembodiments, nucleotides in a nucleic acid (e.g., linked probe molecule)are substituted with specific nucleotide sequences corresponding tospecific nucleotides (“nucleotide representatives”), thereby giving riseto an expanded nucleic acid (e.g., U.S. Pat. No. 6,723,513), and thedetectors hybridize to the nucleotide representatives in the expandednucleic acid, which serves as a base nucleic acid. In such embodiments,nucleotide representatives may be arranged in a binary or higher orderarrangement (e.g., Soni and Meller, Clinical Chemistry 53(11): 1996-2001(2007)). In some embodiments, a nucleic acid is not expanded, does notgive rise to an expanded nucleic acid, and directly serves a basenucleic acid (e.g., a linked probe molecule serves as a non-expandedbase nucleic acid), and detectors are directly contacted with the basenucleic acid. For example, a first detector may hybridize to a firstsubsequence and a second detector may hybridize to a second subsequence,where the first detector and second detector each have detectable labelsthat can be distinguished from one another, and where the signals fromthe first detector and second detector can be distinguished from oneanother when the detectors are disassociated from the base nucleic acid.In certain embodiments, detectors include a region that hybridizes tothe base nucleic acid (e.g., two regions), which can be about 3 to about100 nucleotides in length (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80,85, 90, or 95 nucleotides in length). A detector also may include one ormore regions of nucleotides that do not hybridize to the base nucleicacid. In some embodiments, a detector is a molecular beacon. In someembodiments a detector can be an oligonucleotide species compositionhaving an internal stem-loop that can function as a detectable featurewhen cleaved from the intact oligonucleotide species composition, asdescribed herein. A detector often comprises one or more detectablefeatures independently selected from those described herein. Eachdetectable feature or label can be detected by any convenient detectionprocess capable of detecting a signal generated by each label (e.g.,magnetic, electric, chemical, optical and the like). For example, a CDcamera can be used to detect signals from one or more distinguishablequantum dots linked to a detector.

In certain sequence analysis embodiments, reads may be used to constructa larger nucleotide sequence, which can be facilitated by identifyingoverlapping sequences in different reads and by using identificationsequences in the reads. Such sequence analysis methods and software forconstructing larger sequences from reads are known to the person ofordinary skill (e.g., Venter et al., Science 291: 1304-1351 (2001)).Specific reads, partial nucleotide sequence constructs, and fullnucleotide sequence constructs may be compared between nucleotidesequences within a sample nucleic acid (i.e., internal comparison) ormay be compared with a reference sequence (i.e., reference comparison)in certain sequence analysis embodiments. Internal comparisons sometimesare performed in situations where a sample nucleic acid is prepared frommultiple samples or from a single sample source that contains sequencevariations. Reference comparisons sometimes are performed when areference nucleotide sequence is known and an objective is to determinewhether a sample nucleic acid contains a nucleotide sequence that issubstantially similar or the same, or different, than a referencenucleotide sequence. Sequence analysis can be facilitated by the use ofsequence analysis apparatus and components described above.

Target nucleic acid sequences also can be detected using standardelectrophoretic techniques. Although the detection step can sometimes bepreceded by an amplification step, amplification is not required in theembodiments described herein. Examples of methods for detection andquantification of target nucleic acid sequences using electrophoretictechniques can be found in the art. A non-limiting example is presentedherein. After running a sample (e.g., mixed nucleic acid sample isolatedfrom maternal serum, or amplification nucleic acid species, for example)in an agarose or polyacrylamide gel, the gel may be labeled (e.g.,stained) with ethidium bromide (see, Sambrook and Russell, MolecularCloning: A Laboratory Manual 3d ed., 2001). The presence of a band ofthe same size as the standard control is an indication of the presenceof a target nucleic acid sequence, the amount of which may then becompared to the control based on the intensity of the band, thusdetecting and quantifying the target sequence of interest. In someembodiments, restriction enzymes capable of distinguishing betweenmaternal and paternal alleles may be used to detect and quantify targetnucleic acid species. In certain embodiments, oligonucleotide speciescompositions specific to target nucleic acids (e.g., a specific allele,for example) can be used to detect the presence of the target sequenceof interest. The oligonucleotides can also be used to indicate theamount of the target nucleic acid molecules in comparison to thestandard control, based on the intensity of signal imparted by theoligonucleotide species.

Sequence-specific oligonucleotide species hybridization can be used todetect a particular nucleic acid in a mixture or mixed populationcomprising other species of nucleic acids. Under sufficiently stringenthybridization conditions, the oligonucleotide species (e.g., probes)hybridize specifically only to substantially complementary sequences.The stringency of the hybridization conditions can be relaxed totolerate varying amounts of sequence mismatch. A number of hybridizationformats are known in the art, which include but are not limited to,solution phase, solid phase, or mixed phase hybridization assays. Thefollowing documents provide an overview of the various hybridizationassay formats: Singer et al., Biotechniques 4:230, 1986; Haase et al.,Methods in Virology, pp. 189-226, 1984; Wilkinson, In situHybridization, Wilkinson ed., IRL Press, Oxford University Press,Oxford; and Hames and Higgins eds., Nucleic Acid Hybridization: APractical Approach, IRL Press, 1987.

Hybridization complexes can be detected by techniques known in the art.Nucleic acid probes (e.g., oligonucleotide species) capable ofspecifically hybridizing to a target nucleic acid (e.g., mRNA oramplified DNA) can be labeled by any suitable method, and the labeledprobe used to detect the presence of hybridized nucleic acids. Onecommonly used method of detection is autoradiography, using probeslabeled with 3H, 125I, 35S, 14C, 32P, or the like. The choice ofradioactive isotope depends on research preferences due to ease ofsynthesis, stability, and half-lives of the selected isotopes. Otherlabels include compounds (e.g., biotin and digoxigenin), which bind toantiligands or antibodies labeled with fluorophores, chemiluminescentagents, and enzymes. In some embodiments, probes can be conjugateddirectly with labels such as fluorophores, chemiluminescent agents orenzymes. The choice of label depends on sensitivity required, ease ofconjugation with the probe, stability requirements, and availableinstrumentation.

“Primer extension” polymorphism detection methods also referred toherein as “microsequencing” methods, typically are carried out byhybridizing a complementary oligonucleotide species to a nucleic acidcarrying the polymorphic site. In these methods, the oligonucleotidetypically hybridizes adjacent to the polymorphic site. The term“adjacent” as used in reference to “microsequencing” methods, refers tothe 3′ end of the extension oligonucleotide being sometimes 1 nucleotidefrom the 5′ end of the polymorphic site, often 2 or 3, and at times 4,5, 6, 7, 8, 9, or 10 nucleotides from the 5′ end of the polymorphicsite, in the nucleic acid when the extension oligonucleotide ishybridized to the nucleic acid. The extension oligonucleotide then isextended by one or more nucleotides, often 1, 2, or 3 nucleotides, andthe number and/or type of nucleotides that are added to the extensionoligonucleotide determine which polymorphic variant or variants arepresent. Oligonucleotide extension methods are disclosed, for example,in U.S. Pat. Nos. 4,656,127; 4,851,331; 5,679,524; 5,834,189; 5,876,934;5,908,755; 5,912,118; 5,976,802; 5,981,186; 6,004,744; 6,013,431;6,017,702; 6,046,005; 6,087,095; 6,210,891; and WO 01/20039. Theextension products can be detected in any manner, such as byfluorescence methods (see, e.g., Chen & Kwok, Nucleic Acids Research 25:347-353 (1997) and Chen et al., Proc. Natl. Acad. Sci. USA 94/20:10756-10761 (1997)) or by mass spectrometric methods (e.g., MALDI-TOFmass spectrometry) and other methods described herein. Oligonucleotideextension methods using mass spectrometry are described, for example, inU.S. Pat. Nos. 5,547,835; 5,605,798; 5,691,141; 5,849,542; 5,869,242;5,928,906; 6,043,031; 6,194,144; and 6,258,538.

Microsequencing detection methods often incorporate an amplificationprocess that precedes the extension step. The amplification processtypically amplifies a region from a nucleic acid sample that comprisesthe polymorphic site. Amplification can be carried out utilizing methodsdescribed above, below in the example section or for example using apair of oligonucleotide species compositions described herein, in apolymerase chain reaction (PCR), in which one oligonucleotide speciestypically is complementary to a region 3′ of the polymorphism and theother typically is complementary to a region 5′ of the polymorphism. APCR oligonucleotide species pair may be used in methods disclosed inU.S. Pat. Nos. 4,683,195; 4,683,202, 4,965,188; 5,656,493; 5,998,143;6,140,054; WO 01/27327; and WO 01/27329 for example. PCR oligonucleotidespecies pairs may also be used in any commercially available machinesthat perform PCR, such as any of the GeneAmp® Systems available fromApplied Biosystems.

Whole genome sequencing may also be utilized for discriminating allelesof target nucleic acids (e.g., RNA transcripts or DNA), in someembodiments. Examples of whole genome sequencing methods include, butare not limited to, nanopore-based sequencing methods, sequencing bysynthesis and sequencing by ligation, as described above.

Data Processing

After conducting an enrichment process described herein, enrichednucleic acid or a subset of the enriched nucleic acid or target nucleicacid thereof (collectively enriched nucleic acid”), may be detectedand/or analyzed by any suitable method and any suitable detectiondevice, such as a method or detection device described herein. In someembodiments, one or more target nucleic acids in the enriched nucleicacid may be detected and/or analyzed.

Presence or absence of an outcome can be determined from the detectionand/or analysis results. The term “outcome” as used herein refers to aphenotype indicated by the presence, absence or amount of one or morenucleic acids in the enriched nucleic acid. Non-limiting examples ofoutcomes include presence or absence of a fetus (e.g., pregnancy test),prenatal or neonatal disorder, chromosome abnormality, chromosomeaneuploidy (e.g., trisomy 21, trisomy 18, trisomy 13), cellproliferation condition, other disease condition and the like. Asdescribed herein, algorithms, software, processors and/or machines, forexample, can be utilized to (i) process detection data pertaining toenriched nucleic acid, and/or (ii) identify the presence or absence ofan outcome.

Presence or absence of an outcome may be determined for all samplestested, and in some embodiments, presence or absence of a outcome isdetermined in a subset of the samples (e.g., samples from individualpregnant females). In certain embodiments, an outcome is determined forabout 60, 65, 70, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%, or greater than 99%, ofsamples analyzed in a set. A set of samples can include any suitablenumber of samples, and in some embodiments, a set has about 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 100, 200, 300, 400,500, 600, 700, 800, 900 or 1000 samples, or more than 1000 samples. Theset may be considered with respect to samples tested in a particularperiod of time, and/or at a particular location. The set may beotherwise defined by, for example, gestational age and/or ethnicity. Theset may be comprised of a sample which is subdivided into subsamples orreplicates all or some of which may be tested. The set may comprise asample from the same subject collected at two different times. Incertain embodiments, an outcome is determined about 60% or more of thetime for a given sample analyzed (e.g., about 65, 70, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, 99%, or more than 99% of the time for a given sample). Incertain embodiments, analyzing a higher number of characteristics (e.g.,sequence variations) that discriminate alleles can increase thepercentage of outcomes determined for the samples (e.g., discriminatedin a multiplex analysis). In some embodiments, one or more tissue orfluid samples (e.g., one or more blood samples) are provided by asubject (e.g., pregnant female). In certain embodiments, one or more RNAor DNA samples, or two or more replicate RNA or DNA samples, areisolated from a single tissue or fluid sample, and analyzed by methodsdescribed herein.

Presence or absence of an outcome can be expressed in any suitable form,and in conjunction with any suitable variable, collectively including,without limitation, ratio, deviation in ratio, frequency, distribution,probability (e.g., odds ratio, p-value), likelihood, percentage, valueover a threshold, or risk factor, associated with the presence of aoutcome for a subject or sample. An outcome may be provided with one ormore variables, including, but not limited to, sensitivity, specificity,standard deviation, probability, ratio, coefficient of variation (CV),threshold, score, probability, confidence level, or combination of theforegoing, in certain embodiments.

In certain embodiments, one or more of ratio, sensitivity, specificityand/or confidence level are expressed as a percentage. In someembodiments, the percentage, independently for each variable, is greaterthan about 90% (e.g., about 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%,or greater than 99% (e.g., about 99.5%, or greater, about 99.9% orgreater, about 99.95% or greater, about 99.99% or greater)). Coefficientof variation (CV) in some embodiments is expressed as a percentage, andsometimes the percentage is about 10% or less (e.g., about 10, 9, 8, 7,6, 5, 4, 3, 2 or 1%, or less than 1% (e.g., about 0.5% or less, about0.1% or less, about 0.05% or less, about 0.01% or less)). A probability(e.g., that a particular outcome determined by an algorithm is not dueto chance) in certain embodiments is expressed as a p-value, andsometimes the p-value is about 0.05 or less (e.g., about 0.05, 0.04,0.03, 0.02 or 0.01, or less than 0.01 (e.g., about 0.001 or less, about0.0001 or less, about 0.00001 or less, about 0.000001 or less)).

For example, scoring or a score may refer to calculating the probabilitythat a particular outcome is actually present or absent in asubject/sample. The value of a score may be used to determine forexample the variation, difference, or ratio of amplified nucleicdetectable product that may correspond to the actual outcome. Forexample, calculating a positive score from detectable products can leadto an identification of an outcome, which is particularly relevant toanalysis of single samples.

In certain embodiments, simulated (or simulation) data can aid dataprocessing for example by training an algorithm or testing an algorithm.Simulated data may for instance involve hypothetical various samples ofdifferent concentrations of fetal and maternal nucleic acid in serum,plasma and the like. Simulated data may be based on what might beexpected from a real population or may be skewed to test an algorithmand/or to assign a correct classification based on a simulated data set.Simulated data also is referred to herein as “virtual” data.Fetal/maternal contributions within a sample can be simulated as a tableor array of numbers (for example, as a list of peaks corresponding tothe mass signals of enriched nucleic acids of a reference biomolecule oramplified nucleic acid sequence), as a mass spectrum, as a pattern ofbands on a gel, label intensity, or as a representation of any techniquethat measures mass distribution. Simulations can be performed in mostinstances by a computer program. One possible step in using a simulateddata set is to evaluate the confidence of the identified results, i.e.how well the selected positives/negatives match the sample and whetherthere are additional variations. A common approach is to calculate theprobability value (p-value) which estimates the probability of a randomsample having better score than the selected one. As p-valuecalculations can be prohibitive in certain circumstances, an empiricalmodel may be assessed, in which it is assumed that at least one samplematches a reference sample (with or without resolved variations).Alternatively other distributions such as Poisson distribution can beused to describe the probability distribution.

In certain embodiments, an algorithm can assign a confidence value tothe true positives, true negatives, false positives and false negativescalculated. The assignment of a likelihood of the occurrence of aoutcome can also be based on a certain probability model.

Simulated data often is generated in an in silico process. As usedherein, the term “in silico” refers to research and experimentsperformed using a computer. In silico methods include, but are notlimited to, molecular modeling studies, karyotyping, geneticcalculations, biomolecular docking experiments, and virtualrepresentations of molecular structures and/or processes, such asmolecular interactions.

As used herein, a “data processing routine” refers to a process that canbe embodied in software that determines the biological significance ofacquired data (i.e., the ultimate results of an assay). For example, adata processing routine can determine the amount of each nucleotidesequence species based upon the data collected. A data processingroutine also may control an instrument and/or a data collection routinebased upon results determined. A data processing routine and a datacollection routine often are integrated and provide feedback to operatedata acquisition by the instrument, and hence provide assay-basedjudging methods provided herein.

As used herein, software refers to computer readable programinstructions that, when executed by a computer, perform computeroperations. Typically, software is provided on a program productcontaining program instructions recorded on a computer readable medium,including, but not limited to, magnetic media including floppy disks,hard disks, and magnetic tape; and optical media including CD-ROM discs,DVD discs, magneto-optical discs, and other such media on which theprogram instructions can be recorded.

Different methods of predicting abnormality or normality can producedifferent types of results. For any given prediction, there are fourpossible types of outcomes: true positive, true negative, false positiveor false negative. The term “true positive” as used herein refers to asubject correctly diagnosed as having a outcome. The term “falsepositive” as used herein refers to a subject wrongly identified ashaving a outcome. The term “true negative” as used herein refers to asubject correctly identified as not having a outcome. The term “falsenegative” as used herein refers to a subject wrongly identified as nothaving a outcome. Two measures of performance for any given method canbe calculated based on the ratios of these occurrences: (i) asensitivity value, the fraction of predicted positives that arecorrectly identified as being positives (e.g., the fraction ofnucleotide sequence sets correctly identified by level comparisondetection/determination as indicative of outcome, relative to allnucleotide sequence sets identified as such, correctly or incorrectly),thereby reflecting the accuracy of the results in detecting the outcome;and (ii) a specificity value, the fraction of predicted negativescorrectly identified as being negative (the fraction of nucleotidesequence sets correctly identified by level comparisondetection/determination as indicative of chromosomal normality, relativeto all nucleotide sequence sets identified as such, correctly orincorrectly), thereby reflecting accuracy of the results in detectingthe outcome.

The term “sensitivity” as used herein refers to the number of truepositives divided by the number of true positives plus the number offalse negatives, where sensitivity (sens) may be within the range of0≦sens≦1. Ideally, method embodiments herein have the number of falsenegatives equaling zero or close to equaling zero, so that no subject iswrongly identified as not having at least one outcome when they indeedhave at least one outcome. Conversely, an assessment often is made ofthe ability of a prediction algorithm to classify negatives correctly, acomplementary measurement to sensitivity. The term “specificity” as usedherein refers to the number of true negatives divided by the number oftrue negatives plus the number of false positives, where sensitivity(spec) may be within the range of 0 spec 1. Ideally, methods embodimentsherein have the number of false positives equaling zero or close toequaling zero, so that no subject wrongly identified as having at leastone outcome when they do not have the outcome being assessed. Hence, amethod that has sensitivity and specificity equaling one, or 100%,sometimes is selected.

One or more prediction algorithms may be used to determine significanceor give meaning to the detection data collected under variableconditions that may be weighed independently of or dependently on eachother. The term “variable” as used herein refers to a factor, quantity,or function of an algorithm that has a value or set of values. Forexample, a variable may be the design of a set of amplified nucleic acidspecies, the number of sets of amplified nucleic acid species, percentfetal genetic contribution tested, percent maternal genetic contributiontested, type of outcome assayed, type of sex-linked abnormalitiesassayed, the age of the mother and the like. The term “independent” asused herein refers to not being influenced or not being controlled byanother. The term “dependent” as used herein refers to being influencedor controlled by another. For example, a particular chromosome and atrisomy event occurring for the particular chromosome that results in aviable being are variables that are dependent upon each other.

Any suitable type of method or prediction algorithm may be utilized togive significance to the data of the present technology within anacceptable sensitivity and/or specificity. For example, predictionalgorithms such as Mann-Whitney U Test, binomial test, log odds ratio,Chi-squared test, z-test, t-test, ANOVA (analysis of variance),regression analysis, neural nets, fuzzy logic, Hidden Markov Models,multiple model state estimation, and the like may be used. One or moremethods or prediction algorithms may be determined to give significanceto the data having different independent and/or dependent variables ofthe present technology. And one or more methods or prediction algorithmsmay be determined not to give significance to the data having differentindependent and/or dependent variables of the present technology. Onemay design or change parameters of the different variables of methodsdescribed herein based on results of one or more prediction algorithms(e.g., number of sets analyzed, types of nucleotide species in eachset). For example, applying the Chi-squared test to detection data maysuggest that specific ranges of maternal age are correlated to a higherlikelihood of having an offspring with a specific outcome, hence thevariable of maternal age may be weighed differently verses being weighedthe same as other variables.

In certain embodiments, several algorithms may be chosen to be tested.These algorithms then can be trained with raw data. For each new rawdata sample, the trained algorithms will assign a classification to thatsample (e.g., trisomy or normal). Based on the classifications of thenew raw data samples, the trained algorithms' performance may beassessed based on sensitivity and specificity. Finally, an algorithmwith the highest sensitivity and/or specificity or combination thereofmay be identified.

For a chromosome abnormality, such as aneuploidy for example, chromosomeratio of about 1:1 is expected for a normal, euploid fetus. In someembodiments a ratio of nucleotide sequence species in a set is expectedto be about 1.0:1.0, which can indicate the nucleotide sequence speciesin the set are in different chromosomes present in the same number inthe subject. When nucleotide sequence species in a set are onchromosomes present in different numbers in the subject (for example, intrisomy 21) the set ratio which is detected is lower or higher thanabout 1.0:1.0. Where extracellular nucleic acid is utilized as templatenucleic acid, the measured set ratio often is not 1.0:1.0 (euploid) or1.0:1.5 (e.g., trisomy 21), due to a variety of factors. The expectedmeasured ratio can vary, so long as such variation is substantiallyreproducible and detectable. For example, a particular set might providea reproducible measured ratio (for example of peaks in a massspectrograph) of 1.0:1.2 in a euploid measurement. The aneuploidmeasurement for such a set might then be, for example, 1.0:1.3. The, forexample, 1.3 versus 1.2 measurement is the result of measuring the fetalnucleic acid against a background of maternal nucleic acid, whichdecreases the signal that would otherwise be provided by a “pure” fetalsample, such as from amniotic fluid or from a fetal cell.

In certain embodiments, provided are methods for identifying thepresence or absence of an outcome that comprise: (a) providing a system,wherein the system comprises distinct software modules, and wherein thedistinct software modules comprise a signal detection module, a logicprocessing module, and a data display organization module; (b) detectingsignal information indicating the presence, absence or amount ofenriched nucleic acid; (c) receiving, by the logic processing module,the signal information; (d) calling the presence or absence of anoutcome by the logic processing module; and (e) organizing, by the datadisplay organization model in response to being called by the logicprocessing module, a data display indicating the presence or absence ofthe outcome.

Provided also are methods for identifying the presence or absence of anoutcome, which comprise providing signal information indicating thepresence, absence or amount of enriched nucleic acid; providing asystem, wherein the system comprises distinct software modules, andwherein the distinct software modules comprise a signal detectionmodule, a logic processing module, and a data display organizationmodule; receiving, by the logic processing module, the signalinformation; calling the presence or absence of an outcome by the logicprocessing module; and, organizing, by the data display organizationmodel in response to being called by the logic processing module, a datadisplay indicating the presence or absence of the outcome.

Provided also are methods for identifying the presence or absence of anoutcome, which comprise providing a system, wherein the system comprisesdistinct software modules, and wherein the distinct software modulescomprise a signal detection module, a logic processing module, and adata display organization module; receiving, by the logic processingmodule, signal information indicating the presence, absence or amount ofenriched nucleic acid; calling the presence or absence of an outcome bythe logic processing module; and, organizing, by the data displayorganization model in response to being called by the logic processingmodule, a data display indicating the presence or absence of theoutcome.

By “providing signal information” is meant any manner of providing theinformation, including, for example, computer communication means from alocal, or remote site, human data entry, or any other method oftransmitting signal information. The signal information may be generatedin one location and provided to another location.

By “obtaining” or “receiving” signal information is meant receiving thesignal information by computer communication means from a local, orremote site, human data entry, or any other method of receiving signalinformation. The signal information may be generated in the samelocation at which it is received, or it may be generated in a differentlocation and transmitted to the receiving location.

By “indicating” or “representing” the amount is meant that the signalinformation is related to, or correlates with, for example, the amountof enriched nucleic acid or presence or absence of enriched nucleicacid. The information may be, for example, the calculated dataassociated with the presence or absence of enriched nucleic acid asobtained, for example, after converting raw data obtained by massspectrometry.

Also provided are computer program products, such as, for example, acomputer program products comprising a computer usable medium having acomputer readable program code embodied therein, the computer readableprogram code adapted to be executed to implement a method foridentifying the presence or absence of an outcome, which comprises (a)providing a system, wherein the system comprises distinct softwaremodules, and wherein the distinct software modules comprise a signaldetection module, a logic processing module, and a data displayorganization module; (b) detecting signal information indicating thepresence, absence or amount of enriched nucleic acid; (c) receiving, bythe logic processing module, the signal information; (d) calling thepresence or absence of an outcome by the logic processing module; and,organizing, by the data display organization model in response to beingcalled by the logic processing module, a data display indicating thepresence or absence of the outcome.

Also provided are computer program products, such as, for example,computer program products comprising a computer usable medium having acomputer readable program code embodied therein, the computer readableprogram code adapted to be executed to implement a method foridentifying the presence or absence of an outcome, which comprisesproviding a system, wherein the system comprises distinct softwaremodules, and wherein the distinct software modules comprise a signaldetection module, a logic processing module, and a data displayorganization module; receiving signal information indicating thepresence, absence or amount of enriched nucleic acid; calling thepresence or absence of an outcome by the logic processing module; and,organizing, by the data display organization model in response to beingcalled by the logic processing module, a data display indicating thepresence or absence of the outcome.

Signal information may be, for example, mass spectrometry data obtainedfrom mass spectrometry of a enriched nucleic acid, or of amplifiednucleic acid. As the enriched nucleic acid may be amplified into anucleic acid that is detected, the signal information may be detectioninformation, such as mass spectrometry data, obtained from enrichednucleic acid or stoichiometrically amplified nucleic acid from theenriched nucleic acid, for example. The mass spectrometry data may beraw data, such as, for example, a set of numbers, or, for example, a twodimensional display of the mass spectrum. The signal information may beconverted or transformed to any form of data that may be provided to, orreceived by, a computer system. The signal information may also, forexample, be converted, or transformed to identification data orinformation representing an outcome. An outcome may be, for example, afetal allelic ratio, or a particular chromosome number in fetal cells.Where the chromosome number is greater or less than in euploid cells, orwhere, for example, the chromosome number for one or more of thechromosomes, for example, 21, 18, or 13, is greater than the number ofother chromosomes, the presence of a chromosomal disorder may beidentified.

Also provided is a machine for identifying the presence or absence of anoutcome wherein the machine comprises a computer system having distinctsoftware modules, and wherein the distinct software modules comprise asignal detection module, a logic processing module, and a data displayorganization module, wherein the software modules are adapted to beexecuted to implement a method for identifying the presence or absenceof an outcome, which comprises (a) detecting signal informationindicating the presence, absence or amount of enriched nucleic acid; (b)receiving, by the logic processing module, the signal information; (c)calling the presence or absence of an outcome by the logic processingmodule, wherein a ratio of alleles different than a normal ratio isindicative of a chromosomal disorder; and (d) organizing, by the datadisplay organization model in response to being called by the logicprocessing module, a data display indicating the presence or absence ofthe outcome. The machine may further comprise a memory module forstoring signal information or data indicating the presence or absence ofa chromosomal disorder. Also provided are methods for identifying thepresence or absence of an outcome, wherein the methods comprise the useof a machine for identifying the presence or absence of an outcome.

Also provided are methods identifying the presence or absence of anoutcome that comprises: (a) detecting signal information, wherein thesignal information indicates presence, absence or amount of enrichednucleic acid; (b) transforming the signal information intoidentification data, wherein the identification data represents thepresence or absence of the outcome, whereby the presence or absence ofthe outcome is identified based on the signal information; and (c)displaying the identification data.

Also provided are methods for identifying the presence or absence of anoutcome that comprises: (a) providing signal information indicating thepresence, absence or amount of enriched nucleic acid; (b) transformingthe signal information representing into identification data, whereinthe identification data represents the presence or absence of theoutcome, whereby the presence or absence of the outcome is identifiedbased on the signal information; and (c) displaying the identificationdata.

Also provided are methods for identifying the presence or absence of anoutcome that comprises: (a) receiving signal information indicating thepresence, absence or amount of enriched nucleic acid; (b) transformingthe signal information into identification data, wherein theidentification data represents the presence or absence of the outcome,whereby the presence or absence of the outcome is identified based onthe signal information; and (c) displaying the identification data.

For purposes of these, and similar embodiments, the term “signalinformation” indicates information readable by any electronic media,including, for example, computers that represent data derived using thepresent methods. For example, “signal information” can represent theamount of a enriched nucleic acid or amplified nucleic acid. Signalinformation, such as in these examples, that represents physicalsubstances may be transformed into identification data, such as a visualdisplay that represents other physical substances, such as, for example,a chromosome disorder, or a chromosome number. Identification data maybe displayed in any appropriate manner, including, but not limited to,in a computer visual display, by encoding the identification data intocomputer readable media that may, for example, be transferred to anotherelectronic device (e.g., electronic record), or by creating a hard copyof the display, such as a print out or physical record of information.The information may also be displayed by auditory signal or any othermeans of information communication. In some embodiments, the signalinformation may be detection data obtained using methods to detect aenriched nucleic acid.

Once the signal information is detected, it may be forwarded to thelogic-processing module. The logic-processing module may “call” or“identify” the presence or absence of an outcome.

Provided also are methods for transmitting genetic information to asubject, which comprise identifying the presence or absence of anoutcome wherein the presence or absence of the outcome has beendetermined from determining the presence, absence or amount of enrichednucleic acid from a sample from the subject; and transmitting thepresence or absence of the outcome to the subject. A method may includetransmitting prenatal genetic information to a human pregnant femalesubject, and the outcome may be presence or absence of a chromosomeabnormality or aneuploidy, in certain embodiments.

The term “identifying the presence or absence of an outcome” or “anincreased risk of an outcome,” as used herein refers to any method forobtaining such information, including, without limitation, obtaining theinformation from a laboratory file. A laboratory file can be generatedby a laboratory that carried out an assay to determine the presence orabsence of an outcome. The laboratory may be in the same location ordifferent location (e.g., in another country) as the personnelidentifying the presence or absence of the outcome from the laboratoryfile. For example, the laboratory file can be generated in one locationand transmitted to another location in which the information thereinwill be transmitted to the subject. The laboratory file may be intangible form or electronic form (e.g., computer readable form), incertain embodiments.

The term “transmitting the presence or absence of the outcome to thesubject” or any other information transmitted as used herein refers tocommunicating the information to the subject, or family member, guardianor designee thereof, in a suitable medium, including, withoutlimitation, in verbal, document, or file form.

Also provided are methods for providing to a subject a medicalprescription based on genetic information, which comprise identifyingthe presence or absence of an outcome, wherein the presence or absenceof the outcome has been determined from the presence, absence or amountof enriched nucleic acid from a sample from the subject; and providing amedical prescription based on the presence or absence of the outcome tothe subject.

The term “providing a medical prescription based on prenatal geneticinformation” refers to communicating the prescription to the subject, orfamily member, guardian or designee thereof, in a suitable medium,including, without limitation, in verbal, document or file form.

The medical prescription may be for any course of action determined by,for example, a medical professional upon reviewing the prenatal geneticinformation. For example, the prescription may be for a pregnant femalesubject to undergo an amniocentesis procedure. Or, in another example,the medical prescription may be for the subject to undergo anothergenetic test. In yet another example, the medical prescription may bemedical advice to not undergo further genetic testing.

Also provided are files, such as, for example, a file comprising thepresence or absence of a chromosomal disorder in the fetus of thepregnant female subject, wherein the presence or absence of the outcomehas been determined from the presence, absence or amount of enrichednucleic acid in a sample from the subject.

Also provided are files, such as, for example, a file comprising thepresence or absence of outcome for a subject, wherein the presence orabsence of the outcome has been determined from the presence, absence oramount of enriched nucleic acid in a sample from the subject. The filemay be, for example, but not limited to, a computer readable file, apaper file, or a medical record file.

Computer program products include, for example, any electronic storagemedium that may be used to provide instructions to a computer, such as,for example, a removable storage device, CD-ROMS, a hard disk installedin hard disk drive, signals, magnetic tape, DVDs, optical disks, flashdrives, RAM or floppy disk, and the like.

The systems discussed herein may further comprise general components ofcomputer systems, such as, for example, network servers, laptop systems,desktop systems, handheld systems, personal digital assistants,computing kiosks, and the like. The computer system may comprise one ormore input means such as a keyboard, touch screen, mouse, voicerecognition or other means to allow the user to enter data into thesystem. The system may further comprise one or more output means such asa CRT or LCD display screen, speaker, FAX machine, impact printer,inkjet printer, black and white or color laser printer or other means ofproviding visual, auditory or hardcopy output of information.

The input and output means may be connected to a central processing unitwhich may comprise among other components, a microprocessor forexecuting program instructions and memory for storing program code anddata. In some embodiments the methods may be implemented as a singleuser system located in a single geographical site. In other embodimentsmethods may be implemented as a multi-user system. In the case of amulti-user implementation, multiple central processing units may beconnected by means of a network. The network may be local, encompassinga single department in one portion of a building, an entire building,span multiple buildings, span a region, span an entire country or beworldwide. The network may be private, being owned and controlled by theprovider or it may be implemented as an Internet based service where theuser accesses a web page to enter and retrieve information.

The various software modules associated with the implementation of thepresent products and methods can be suitably loaded into the a computersystem as desired, or the software code can be stored on acomputer-readable medium such as a floppy disk, magnetic tape, or anoptical disk, or the like. In an online implementation, a server and website maintained by an organization can be configured to provide softwaredownloads to remote users. As used herein, “module,” includinggrammatical variations thereof, means, a self-contained functional unitwhich is used with a larger system. For example, a software module is apart of a program that performs a particular task. Thus, provided hereinis a machine comprising one or more software modules described herein,where the machine can be, but is not limited to, a computer (e.g.,server) having a storage device such as floppy disk, magnetic tape,optical disk, random access memory and/or hard disk drive, for example.

The present methods may be implemented using hardware, software or acombination thereof and may be implemented in a computer system or otherprocessing system. An example computer system may include one or moreprocessors. A processor can be connected to a communication bus. Thecomputer system may include a main memory, sometimes random accessmemory (RAM), and can also include a secondary memory. The secondarymemory can include, for example, a hard disk drive and/or a removablestorage drive, representing a floppy disk drive, a magnetic tape drive,an optical disk drive, memory card etc. The removable storage drivereads from and/or writes to a removable storage unit in a well-knownmanner. A removable storage unit includes, but is not limited to, afloppy disk, magnetic tape, optical disk, etc. which is read by andwritten to by, for example, a removable storage drive. As will beappreciated, the removable storage unit includes a computer usablestorage medium having stored therein computer software and/or data.

In alternative embodiments, secondary memory may include other similarmeans for allowing computer programs or other instructions to be loadedinto a computer system. Such means can include, for example, a removablestorage unit and an interface device. Examples of such can include aprogram cartridge and cartridge interface (such as that found in videogame devices), a removable memory chip (such as an EPROM, or PROM) andassociated socket, and other removable storage units and interfaceswhich allow software and data to be transferred from the removablestorage unit to a computer system.

The computer system may also include a communications interface. Acommunications interface allows software and data to be transferredbetween the computer system and external devices. Examples ofcommunications interface can include a modem, a network interface (suchas an Ethernet card), a communications port, a PCMCIA slot and card,etc. Software and data transferred via communications interface are inthe form of signals, which can be electronic, electromagnetic, opticalor other signals capable of being received by communications interface.These signals are provided to communications interface via a channel.This channel carries signals and can be implemented using wire or cable,fiber optics, a phone line, a cellular phone link, an RF link and othercommunications channels. Thus, in one example, a communicationsinterface may be used to receive signal information to be detected bythe signal detection module.

In a related aspect, the signal information may be input by a variety ofmeans, including but not limited to, manual input devices or direct dataentry devices (DDEs). For example, manual devices may include,keyboards, concept keyboards, touch sensitive screens, light pens,mouse, tracker balls, joysticks, graphic tablets, scanners, digitalcameras, video digitizers and voice recognition devices. DDEs mayinclude, for example, bar code readers, magnetic strip codes, smartcards, magnetic ink character recognition, optical characterrecognition, optical mark recognition, and turnaround documents. In oneembodiment, an output from a gene or chip reader my serve as an inputsignal.

EXAMPLES

The examples set forth below illustrate certain embodiments and do notlimit the technology.

Example 1: Size Selection Based DNA Extraction

FIG. 1 illustrates the results of size selection based DNA extractionusing a 1-kilobase (kb) ladder and various salt concentrations. The DNAis first bound to a silica dioxide solid support at a high saltconcentration of guanidine thiocyanate to bind all nucleic acids in anucleic acid composition (FIG. 1A). Different salt concentrations wereused to select for different sizes of nucleic acids from the beads.Nucleic acids within specific size ranges (e.g., 100 to 200 bp, 200 to300 bp and the like) can be enriched or extracted based on the use ofparticular salt concentrations, as described below and presented inTable 1 (see Example 3).

To identify salt concentrations useful for eluting nucleic acids ofvarying sizes from the solid support beads, a titration of differentsalt concentrations was used to remove the smaller nucleic acidfragments of a 1-kb ladder from the beads and determine the saltconcentrations at which different sized, bound, nucleic acids wereeluted from the beads (FIG. 1B). This was accomplished by first bindingsubstantially all of the nucleic acid composition (e.g., the 1-kbladder) to the beads, then applying different salt concentrations (0.25MFIG. 1B lanes 1-2, 0.375M FIG. 1B lanes 3-4, and 0.5M NaCl FIG. 1B lanes5-6) to the same beads to extract (e.g., elute) the smaller fragments,leaving behind the larger fragments. The larger fragments were elutedoff the beads and analyzed in an Agilent BioAnalyzer. For selection ofsmaller size DNA fragments, the titration of different salts wasperformed as above (e.g., 0.25M FIG. 10 lanes 1-2, 0.375M FIG. 10 lanes3-4, and 0.5M NaCl FIG. 10 lanes 5-6) to remove the smaller fragmentsfrom the beads. The supernatant containing the eluted fragments wasremoved to a new tube, contacted with new beads in the presence of ahigh concentration of guanidine thiocyanate chaotropic salt (e.g.,binding or adsorbing), followed by extracting and concentrating thesmall fragments (FIG. 10).

In some embodiments, the method can be employed to facilitate sizeselective separation of nucleic acid ranges useful for further analysisusing many different methods, such as enrichment of fetal or diseasenucleic acids from a background of maternal or healthy nucleic acids orlibrary preparations for sequencing, for example. Figure specificmethods are further described below.

FIG. 1A

-   -   1. 1 μg of 1-kb ladder was added to a final concentration of        1.4M GuSCN, 33% ETOH and sufficient silica dioxide magnetic        beads to bind all of the DNA. The mixture was shaken at 800        revolutions per minute (RPM) at room temperature for 10 minutes.    -   2. The mixture was placed in a magnetic field and the        supernatant was removed.    -   3. 400 μl of a 90% ETOH solution was added to the beads, placed        in a magnetic field and removed twice to wash out the        inhibitors.    -   4. The beads were allowed to air dry 5 minutes to remove the        ETOH wash.    -   5. 10 μl of DEPC H₂O was added the beads to elute the DNA.    -   6. 1.5 μl of the sample was run on an Agilent BioAnalyzer DNA        1000 chip.

FIG. 1B

-   -   1. 1 μg of 1-kb ladder was added to a final concentration of        1.4M GuSCN, 33% ETOH and sufficient silica dioxide magnetic        beads to bind all of the DNA. The mixture was shaken at 800        revolutions per minute (RPM) at room temperature for 10 minutes.    -   2. The mixture was placed in a magnetic field and the        supernatant was removed.    -   3. 400 μl of a 90% ETOH solution was added to the beads, placed        in a magnetic field and removed twice to wash out the        inhibitors.    -   4. The beads were allowed to air dry 5 minutes to remove the        ETOH wash.    -   5. The beads are added to a solution containing 10% Crowding        Agent and salt at a final concentration of 0.5M, 0.375M or 0.25M        NaCl, and incubated at 45° C. for 10 minutes.    -   6. The mixture was placed in a magnetic field and the        supernatant was removed.    -   7. 400 μl of a 90% ETOH solution was added to the beads, placed        in a magnetic field and removed twice to wash out the        inhibitors.    -   8. The beads were allowed to air dry 5 minutes to remove the        ETOH wash.    -   9. 10 μl of DEPC H₂O was added the beads to elute the DNA.    -   10. 1.5 μl of the sample was run on an Agilent BioAnalyzer DNA        1000 chip.

FIG. 10

-   -   1. 1 μg of 1-kb ladder was added to a final concentration of        1.4M GuSCN, 33% ETOH and sufficient silica dioxide magnetic        beads to bind all of the DNA. The mixture was shaken at 800        revolutions per minute (RPM) at room temperature for 10 minutes.    -   2. The mixture was placed in a magnetic field and the        supernatant was removed.    -   3. 400 μl of a 90% ETOH solution was added to the beads, placed        in a magnetic field and removed twice to wash out the        inhibitors.    -   4. The beads were allowed to air dry 5 minutes to remove the        ETOH wash.    -   5. The beads are added to a solution containing 10% crowding        agent and salt at a final concentration of 0.5M, 0.375M or 0.25M        NaCl, and incubated at 45° C. for 10 minutes.    -   6. The mixture was placed in a magnetic field and the        supernatant was removed.    -   7. The supernatant was added to a solution containing a final        concentration of 1.4M GuSCN and 33% ETOH and sufficient silica        dioxide magnetic beads to bind all of the DNA.    -   8. 400 μl of a 90% ETOH solution was added to the beads, placed        in a magnetic field and removed twice to wash out the        inhibitors.    -   9. The beads were allowed to air dry 5 minutes to remove the        ETOH wash.    -   10. 10 μl of DEPC H₂O was added the beads to elute the DNA.    -   11. 1.5 μl of the sample was run on an Agilent BioAnalyzer DNA        1000 chip.

Table A shows ratios of relatively small to relatively large nucleicacid eluted from the solid supports (200 bp or less and the 300 bp orless fractions), and ratios of relatively large to relatively smallnucleic acid associated with solid supports (greater than 200 bp andgreater than 300 bp fractions) for various dissociation conditions.

TABLE A Base Pairs 0.5M NaCl/ 0.375M NaCl/ 0.25M NaCl/ 0.5M NaCl/ 0.375MNaCl/ 0.25M NaCl/ for a 1 kb 10% 10% 10% 18% 18% 18% Ladder PEG8000PEG8000 PEG8000 PEG8000 PEG8000 PEG8000 200 bp or less 2.3 1.6 0.23 0.70.9 0.25 >200 bp 16 68 109 1.4 1.2 3.8 300 bp or less 3.6 3.48 0.53 0.91 0.3 >300 bp 6.4 28 62 0.7 1 3.48

Example 2: Isolation of DNA in a Plasma Sample

The methods and compositions provided herein can be used to selectivelyenrich and/or extract DNA based on its size in a maternal plasma sample(see FIG. 2). Whole blood was collected from a pregnant female andcentrifuged to obtain the plasma fraction containing cell free DNA fromboth mother and fetus. Extracellular maternal nucleic acid ranges insize distribution from about 50 bp to about 800 bp, while fetal DNAranges from about 50 bp to about 300 bp (Chan et al (2004) and Li et al(2004). The difference in size ranges at the upper end of the nucleicacid fragment size range, as seen between maternal and fetal nucleicacids, allows for size specific enrichment of fetal nucleic acid. Theresults illustrated in FIG. 2 show a 30% enrichment of a male fetal DNAfrom maternal DNA by selecting for sizes 300 bp, with the greatestenrichment seen 200 bp. Without enrichment, fetal DNA is 9% of the totalnucleic acid isolated from maternal plasma. Enrichment was performed byusing three different salt titrations 0.375M NaCl/10% PEG, 0.5M NaCl/10%PEG, and 1M NaCl/10% PEG, which selects for less than 500 base pairs,less than 400 base pairs, and less than 300 base pairs, respectively. InFIG. 2, 30% enrichment (e.g., 100%−(9%/13%)) of the male fetal DNA isachieved by selecting for 300 bp and lower.

1. Protein Denaturation and Protein Digestion

An aqueous buffer with a pH in the range of about 5 to about 8 andcontaining; a low concentration of chaotropic salt (e.g., less than 30%solution (weight per volume, or w/v), for example), a detergent at 5-20%w/v, 1-50 mM EDTA, and protease or proteinase k at 5-100 mg/ml was addedto a blood plasma sample to denature proteins and inactivate nucleases.The solution was mixed thoroughly, and incubated according to therequirements for the chosen protein degrading enzyme (e.g., 30 minutesat 55° C., for example).

2. Binding of Nucleic Acids

The sample was contacted with a solid support (e.g., beads, for example)and adjusted to a final concentration of 1.4M GuSCN and 33% ETOH. Themixture was incubated with rotation at room temperature for 20 minutes.

3. Separation of the Solid Support from the Solution

The beads were separated from the supernatant by centrifugation ormagnetic field.

4. Washing the Beads to Remove Inhibitors

The beads were resuspended in 800 μl of 90% ETOH, placed in a magneticfield and the wash solution removed. Washes were typically performedtwice to ensure removal of substantially all PCR inhibitors.

5. Size Selection of Nucleic Acids Isolated from Plasma Samples

The washed beads, to which total serum DNA was bound, were resuspendedin a solution containing 10% Crowding Agent and a specific saltconcentration chosen according to the specific size range of nucleicacids desired. The beads were mixed thoroughly, and incubated for 10minutes at 45° C.

6. Separation of Eluted Nucleic Acids Fragments and Binding to New Beads

The beads are placed in a magnetic field and the supernatant wascollected and transferred to a new tube. The concentration of thesolution was adjusted to 1.4M GuSCN and 33% ETOH to bind the elutedfragments (e.g., small fragments) contained in the supernatant solution.

7. Washing

The beads were resuspended in 800 μl of 90% ETOH, placed in a magneticfield and the wash solution removed. Washes were typically performedtwice to ensure removal of substantially all PCR inhibitors. The beadswith the larger fragments were also washed using the same conditions, toallow a comparison of the size ranges eluted under the various saltconditions. The larger fragments can also be analyzed further. All beadswere subjected to air drying to remove any remaining alcohol that mightinhibit further analysis.

8. Elution

The target nucleic acids were eluted from the solid support by additionof a sufficient quantity of sterile water or aqueous buffered solution(e.g., 1×TE pH 7-8.5). The elution can be performed at ambienttemperature or by exposing to heat. The eluate was collected andprepared for further analysis

9. Analysis of the Target DNA

An aliquot of the eluted DNA is subjected to PCR using multiplexedprimers and a reference target of known copy number and sequence, aslisted in Table 2 (presented below in Example 4). The PCR conditionsused to amplify the target nucleic acid were; 50° C. for 3 minutes, 93°C. for 10 minutes, 45 cycles of 93° C. for 5 seconds, 60° C. for 30seconds and 72° C. for 1 minute and 15 seconds, followed by a hold at72° C. for 10 minutes. The PCR products were analyzed with MassArrayspectrometry, using qGE protocols to determine copy number ratio of malefetus Y chromosome DNA to total DNA using PLAC 4 genes and RhD loci. Thedata illustrates successful enrichment and extraction of small fragmentscontaining fetal nucleic acids.

Table 1 illustrates the percent recovery of specific size fractions ofnucleic acid, using specific dissociabon conditions from the totalnucleic acid isolated from a substantially cell free sample

Large Fragments 0.5M 0.375M 0.25M 0.5M 0.375M 0.25M 0.5M NaCl/ NaCl/NaCl/ NaCl/ NaCl/ NaCl/ NaCl/ Base 10% 10% 10% 18% 18% 18% 10% PairsPEG8000 PEG8000 PEG8000 PEG8000 PEG8000 PEG8000 PEG8000 for a LargeLarge Large Large Large Large Small 1 kg Frag- Frag- Frag- Frag- Frag-Frag- Frag- Ladder ments ments ments ments ments ments ments  100 bp  0% 0%  0%  0%  0%  0% 100%  200 bp  0%  0%  0%  51%  0%  0% 100%  300 bp 0%  13%  0%  60%  11%  0% 100%  400 bp  39%  50%  0%  84%  27%  12% 61%  500 bp  78%  80%  0% 100%  34%  17%  22%  600 bp 100%  97%  0%100%  34%  27%  0%  700 bp 100% 100%  0% 100%  51%  44%  0%  800 bp 100%100%  0% 100%  61%  52%  0%  900 bp 100% 100%  21% 100%  72%  59%  0%1000 bp 100% 100%  35% 100%  80%  65%  0% 1200 bp 100% 100% 100% 100%100% 114%  0% 1300 bp 100% 100%  77% 100%  22%  18%  0% Small Fragments0.375M 0.25M 0.5M 0.375M 0.25M NaCl/ NaCl/ NaCl/ NaCl/ NaCl/ Base 10%10% 18% 18% 18% Pairs PEG8000 PEG8000 PEG8000 PEG8000 PEG8000 for aSmall Small Small Small Small 1 kg Frag- Frag- Frag- Frag- Frag- Ladderments ments ments ments ments  100 bp 100% 100% 100% 100% 100%  200 bp100% 100%  49% 100% 100%  300 bp  87% 100%  40%  89% 100%  400 bp  50%100%  16%  73%  88%  500 bp  20% 100%  0%  66%  83%  600 bp  3% 100%  0% 66%  73%  700 bp  0% 100%  0%  49%  56%  800 bp  0% 100%  0%  39%  48% 900 bp  0%  79%  0%  28%  41% 1000 bp  0%  65%  0%  20%  35% 1200 bp 0%  0%  0%  0%  0% 1300 bp  0%  23%  0%  78%  82%

TABLE 2provides the sequences of oligonucleotides, and probes used to analyze the nucleic acids recovered using the methods andcompositions described herein. SNP_ID Forward Primers Reverse PrimersProbe RhD Ex4 ACGTTGGATGGACTATCAGGGCTTGCCCCGACGTTGGATGTGCGAACACGTAGATGTGCA cTGCAGACAGACTACCAC ATGAAC RhD Ex7-D1ACGTTGGATGAGCTCCATCATGGGCTACAAC ACGTTGGATGTTGCCGGCTCCGACGGTATCCTTGCTGGGTCTGCTTGG AGAGATCA SRY ACGTTGGATGAGATGGCTCTAGAGAATCCCACGTTGGATGGCATTTTCCACTGGTATCCC CCAGAATGCGAAACTC P4_rs8130833NEACGTTGGATGTATAGAACCATGTTTAGG ACGTTGGATGACCATTTGGGTTAAATACTTTGGGTTAAATACAAGTT AGA P4_rs4818219Cur ACGTTGGATGTCTGGGACTAGTACCCAAAGACGTTGGATGAAAGCCACTGACAAGCAGAC GGGATGGCTTGCGCAGT G P4_rs8130833ACGTTGGATGACCATTTGGGTTAAATAC ACGTTGGATGTATAGAACCATGTTTAGGGCATGTTTAGGCCAGATA Competitor TATTCG P4_rs4818219ACGTTGGATGTCTGGGACTAGTACCCAAAG ACGTTGGATGAAAGCCACTGACAAGCAGACCCAAAGCACCTAGCTCTC Competitor C Control ACGTTGGATGAGTGGACTCCAGGTAAGATGACGTTGGATGGATGGCAGCCTGAATATGTC TCGATTCCTAGAACTGTT RhD Ex5-D2ACGTTGGATGAATCGAAAGGAAGAATGCCG ACGTTGGATGCTGAGATGGCTGTCACCACGCCCGTGTTCAACACCTAC TATGCT X/Y TCGACCCGGAGCACGTTGGAACACTCCATGACTCCAACCCTCGACCCGGAGCACGTTGGAGCTGGTAGGG CCCAGCAGCCAAACCTCC CTGCTGGGC CTCSRY Competitor GATCAGAGGCGCAAGATGGCTCTAGAGAATCCCAGAATGCGAAAC TemplateTCTGAGATCAGCAAGCAGCTGGGATACCAGTGGAAAATGCTTACT GAAGCCGARHD-Ex5 Competitor AAGGATGACCCTGAGATGGCTGTCACCACGCTGACTGCTATAGCATemplate TAGTAGGTGTTGAACACGGCATTCTTCCTTTCGATTGGACTTCTCARHD-Ex4 Competitor TTCTCCAAGGACTATCAGGGCTTGCCCCGGACGACACTCACTGCT TmplateCTTACTGGGTTTTATTGCAGACAGACTACCACATGAACGTGATGCA CATCTACGTGTTCGCAGRHD-Ex7 Competitor ATTCCCCACAGCTCCATCATGGGCTACAACTAGCTTGCTGGGTCTTemplate GCTTGGAGAGATCAACTTTGTGCTGCTGGTGCTTGATACCGTCGGAGCCGGCAATGGCATGTG rs8130833 CompetitorAACACCATTTGGGTTAAATACACAAGTCTTGTCGAATATATCTGGC TemplateCTAAACATGGTTCTATATACT rs4818219 CompetitorAGAAAAGCCACTGACAAGCAGACAGAATACTACTGTCAATATAGGA TmplateGAGCTAGGTGCTTTGGGTACTAGTCCCAGAGCT

Example 3: Embodiments

Provided hereafter are certain non-limiting embodiments. Not allembodiments are sequentially labeled.

A1. A method for enriching relatively short nucleic acid from a nucleicacid composition, which comprises:

-   -   (a) contacting nucleic acid of a nucleic acid composition with a        solid phase under association conditions, wherein:        -   (i) the nucleic acid of the nucleic acid composition            comprises relatively short nucleic acid and relatively long            nucleic acid,        -   (ii) the relatively short nucleic acid is about 300 base            pairs or less, and        -   (iii) the relatively long nucleic acid is larger than about            300 base pairs;        -   whereby the relatively short nucleic acid and the relatively            long nucleic acid are associated with the solid phase;    -   (b) introducing the solid phase after (a) to dissociation        conditions that comprise a volume exclusion agent and a salt,        wherein:        -   (i) the salt is not a chaotropic salt, and        -   (ii) the relatively short nucleic acid preferentially            dissociates from the solid phase under the dissociation            conditions as compared to the relatively long nucleic,            thereby yielding dissociated nucleic acid; and    -   (c) separating the dissociated nucleic acid from the solid        phase, whereby the relatively short nucleic acid is enriched in        the dissociated nucleic acid relative to in the nucleic acid        composition.

A1.1 The method of embodiment A1, wherein the nucleic acid compositionis a biological composition.

A1.2 The method of embodiment A1.1, wherein the biological compositionis a substantially cell-free biological composition.

A1.3 The method of embodiment A1.1, wherein the nucleic acid iscell-free nucleic acid.

A2. The method of embodiment A1.2, wherein the substantially cell-freebiological composition is from a pregnant female.

A3. The method of embodiment A2, wherein the pregnant female is in thefirst trimester of pregnancy.

A4. The method of any one of embodiments A1.2-A3, wherein thesubstantially cell-free biological composition is blood serum.

A5. The method of any one of embodiments A1.2-A3, wherein thesubstantially cell-free biological composition is blood plasma.

A6. The method of any one of embodiments A1.2-A3, wherein thesubstantially cell-free biological composition is urine.

A6.5. The method of any one of embodiments A1-A6, wherein the solidphase is a collection of particles.

A7. The method of embodiment A6.5, wherein the particles comprisesilica.

A7.1. The method of embodiment A7, wherein the silica comprises silicadioxide.

A8. The method of embodiment A7 or A7.1, wherein the particles furthercomprise an agent that confers a paramagnetic property to the particles.

A9. The method of embodiment A8, wherein the agent comprises a metal.

A9.1. The method of embodiment A9, wherein the agent is a metal oxide.

A10. The method of any one of embodiments A1-A9.1, wherein the solidphase does not comprise a functional group that interacts with thenucleic acid.

A11. The method of embodiment A10, wherein the solid phase does notcomprise a carboxy functional group.

A11.1. The method of any one of embodiments A1-A11, wherein the solidphase has a net charge.

A11.2. The method of embodiment A11.1, wherein the net charge ispositive.

A11.3. The method of embodiment A11.1, wherein the net charge isnegative.

A12. The method of any one of embodiments A1-A11.3, wherein thedissociated nucleic acid comprises deoxyribonucleic acid (DNA).

A13. The method of any one of embodiments A1-A12, wherein thedissociated nucleic acid comprises ribonucleic acid (RNA).

A14. The method of any one of one of embodiments A1-A12, wherein thedissociated nucleic acid consists essentially of DNA.

A15. The method of any one of embodiments A1-A11.3, wherein thedissociated nucleic acid consists essentially of RNA.

B1. The method of any one of embodiments A1-A15, wherein the associationconditions comprise a C1-C6 alkyl alcohol.

B2. The method of any one of embodiments A1-A15, wherein the associationconditions consist essentially of a C1-C6 alkyl alcohol.

B3. The method of any one of embodiments A1-A15, wherein the associationconditions do not comprise a C1-C6 alkyl alcohol.

B4. The method of any one of embodiments B1-B3, wherein the alcoholcomprises ethanol.

B5. The method of any one of embodiments A1-A15, wherein the associationconditions comprise a salt.

B6. The method of any one of embodiments A1-A15, wherein the associationconditions consist essentially of a salt.

B7. The method of any one of embodiments A1-A15, wherein the associationconditions do not comprise a salt.

B8. The method of any one of embodiments B5-B7, wherein the saltcomprises a chaotropic salt, an ionic salt or combination thereof.

B9. The method of any one of embodiments A1-A15, wherein the associationconditions comprise a volume exclusion agent.

B10. The method of any one of embodiments A1-A15, wherein theassociation conditions consist essentially of a volume exclusion agent.

B11. The method of any one of embodiments A1-A15, wherein theassociation conditions do not comprise a volume exclusion agent.

B12. The method of any one of embodiments B9-B11, wherein the volumeexclusion agent comprises a polyalkyl glycol, dextran, Ficoll, polyvinylpyrollidone or combination thereof.

B13. The method of any one of embodiments A1-A15 and B1-B12, wherein therelatively short nucleic acid is about 200 base pairs or less and therelatively long nucleic acid is larger than about 200 base pairs.

B14. The method of embodiment B13, wherein the relatively short nucleicacid is about 50 to about 180 base pairs.

B15. The method of any one of embodiments A1-A15 and B1-B14, whereinabout 30% to about 90% of the nucleic acid of the nucleic acidcomposition associates with the solid phase.

B16. The method of embodiment B15, wherein about 60% of the nucleic acidof the nucleic acid composition associates with the solid phase.

B17. The method of any one of embodiments A1-A15 and B1-B16, whichfurther comprises washing the solid phase after (a).

B17.1. The method of embodiment B17, wherein the solid phase is washedunder conditions that remove material of the nucleic acid compositionnot associated with the solid phase from the solid phase.

B17.2. The method of embodiment B17, wherein the solid phase is washedunder conditions that dissociate any non-nucleic acid material of thenucleic acid composition from the solid phase.

C1. The method of any one of embodiments A1-A15 and B1-B17, wherein thesalt comprises an ionic salt.

C2. The method of embodiment C1, wherein the ionic salt is sodiumchloride.

C3. The method of embodiment C1 or C2, wherein the dissociationconditions comprise about 0.25M to about 0.5M of the ionic salt.

C4. The method of any one of embodiments A1-A15, B1-B17 and C1-C3,wherein the volume exclusion agent comprises a polyalkyl alcohol,dextran, Ficoll, polyvinyl pyrollidone or combination thereof.

C5. The method of embodiment C4, wherein the polyalkyl alcohol ispolyethylene glycol (PEG).

C6. The method of embodiment C5, wherein the PEG is PEG 8000

C7. The method of embodiment C5 or C6, wherein the dissociationconditions comprise about 10% PEG.

C8. The method of any one of embodiments A1-A15, B1-B17 and C1-C7,wherein the salt and the volume exclusion agent are present in thedissociation conditions at concentrations according to Table 1.

C9. The method of any one of embodiments A1-A15, B1-B17 and C1-C8,wherein the relatively short nucleic acid preferentially dissociatesfrom the solid phase under the dissociation conditions as compared tothe relatively long nucleic acid at a ratio of about 1.05 to about 5relatively short nucleic acid to relatively long nucleic acid.

C11. The method of any one of embodiments A1-A15, B1-B17 and C1-C10,wherein the relatively short nucleic acid is enriched about 10% to about45% in the dissociated nucleic acid relative to in the nucleic acidcomposition.

C12. The method of any one of embodiments A1-A15, B1-B17 and C1-C11,wherein the solid phase is paramagnetic and the dissociated nucleic acidis separated from the solid phase by a magnet.

C13. The method of any one of embodiments A1-A15, B1-B17 and C1-C11,wherein the solid phase is separated from the dissociated nucleic acidby centrifugation.

C14. The method of any one of embodiments A1-A15, B1-B17 and C1-C13,wherein the solid phase is separated from the dissociated nucleic acidby transferring the dissociated nucleic acid to an environment that doesnot contain the solid phase used in (a) of embodiment A1.

C15. The method of any one of embodiments A1-A15, B1-B17 and C1-C13,wherein the solid phase is separated from the dissociated nucleic acidby transferring the solid phase to an environment that does not containthe dissociated nucleic acid.

C16. The method of embodiment C14 or C15, wherein the environment is avessel.

C17. The method of any one of embodiments A1-A15, B1-B17 and C1-C16,which further comprises associating the dissociated nucleic acid to asecond solid phase.

C18. The method of embodiment C17, which further comprises dissociatingthe dissociated nucleic acid from the second solid phase, therebyreleasing the dissociated nucleic acid from the second solid phase.

D1. The method of any one of embodiments A1-A15, B1-B17 and C1-C18,which further comprises analyzing the dissociated nucleic acid and/ornucleic acid associated with the solid phase after (c) by massspectrometry.

D2. The method of any one of embodiments A1-A15, B1-B17 and C1-C18,which further comprises contacting the dissociated nucleic acid and/ornucleic acid associated with the solid phase after (c) with anoligonucleotide that hybridizes to the dissociated nucleic acid and isextended under extension conditions, thereby yielding extendedoligonucleotide.

D3. The method of any one of embodiments A1-A15, B1-B17 and C1-C18,which further comprises amplifying the dissociated nucleic acid and/orthe nucleic acid associated with the solid phase after (c), therebyyielding amplified product.

D4. The method of embodiment D3, which further comprises contacting theamplified product with an oligonucleotide that hybridizes to theamplified product and is extended under extension conditions, therebyyielding extended oligonucleotide.

D5. The method of any one of embodiments D2-D4, which further comprisesanalyzing the extended oligonucleotide or the amplified product.

D6. The method of embodiment D5, wherein the extended oligonucleotide orthe amplified product is analyzed by mass spectrometry.

D7. The method of any one of embodiments A1-A15, B1-B17, C1-C18 andD1-D6, which further comprises detecting the presence or absence offetal nucleic acid.

D8. The method of embodiment D7, which comprises detecting the presenceor absence of a fetal-specific nucleotide sequence.

D9. The method of embodiment D8, wherein the fetal-specific nucleotidesequence is a Y-chromosome sequence.

D10. The method of embodiment D8, wherein the fetal-specific nucleotidesequence is a mRNA sequence.

D11. The method of any one of embodiments A1-A15, B1-B17, C1-C18 andD1-D10, which further comprises detecting the presence or absence of aprenatal disorder.

D12. The method of embodiment D11, wherein the prenatal disorder is achromosome abnormality.

D13. The method of embodiment D12, wherein the chromosome abnormality isa trisomy.

D14. The method of embodiment D13, wherein the trisomy is trisomy 21,trisomy 18, trisomy 13 or combination thereof.

D15. The method of any one of embodiments A1-A15, B1-B17, C1-C18 andD1-D14, which further comprises detecting the presence or absence of acell proliferation disorder.

D16. The method of embodiment D15, wherein the cell proliferationdisorder is a cancer.

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. Citationof the above patents, patent applications, publications and documents isnot an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents.

Modifications may be made to the foregoing without departing from thebasic aspects of the technology. Although the technology has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments specifically disclosed in thisapplication, yet these modifications and improvements are within thescope and spirit of the technology.

The technology illustratively described herein suitably may be practicedin the absence of any element(s) not specifically disclosed herein.Thus, for example, in each instance herein any of the terms“comprising,” “consisting essentially of,” and “consisting of” may bereplaced with either of the other two terms. The terms and expressionswhich have been employed are used as terms of description and not oflimitation, and use of such terms and expressions do not exclude anyequivalents of the features shown and described or portions thereof, andvarious modifications are possible within the scope of the claimedtechnology. The term “a” or “an” can refer to one of or a plurality ofthe elements it modifies (e.g., “a reagent” can mean one or morereagents) unless it is contextually clear either one of the elements ormore than one of the elements is described. The term “about” as usedherein refers to a value within 10% of the underlying parameter (i.e.,plus or minus 10%), and use of the term “about” at the beginning of astring of values modifies each of the values (i.e., “about 1, 2 and 3”refers to about 1, about 2 and about 3). For example, a weight of “about100 grams” can include weights between 90 grams and 110 grams. Further,when a listing of values is described herein (e.g., about 50%, 60%, 70%,80%, 85% or 86%) the listing includes all intermediate and fractionalvalues thereof (e.g., 54%, 85.4%). Thus, it should be understood thatalthough the present technology has been specifically disclosed byrepresentative embodiments and optional features, modification andvariation of the concepts herein disclosed may be resorted to by thoseskilled in the art, and such modifications and variations are consideredwithin the scope of this technology.

Certain embodiments of the technology are set forth in the claim(s) thatfollow(s).

1. (canceled)
 2. A method for enriching relatively short nucleic acidfrom a nucleic acid composition, which comprises: (a) contactingcell-free nucleic acid from a substantially cell-free biologicalcomposition with a solid phase under association conditions, wherein:(i) the cell-free nucleic acid of the substantially cell-free biologicalcomposition comprises relatively short nucleic acid and relatively longnucleic acid, (ii) the relatively short nucleic acid is about 300 basepairs or less; (iii) the relatively long nucleic acid is larger thanabout 300 base pairs, and (iv) the association conditions do not includepolyethylene glycol; (b) introducing the solid phase after (a) todissociation conditions that comprise a salt, wherein: (i) the salt isnot a chaotropic salt, and (ii) the relatively short nucleic aciddissociates from the solid phase under the dissociation conditions,thereby yielding dissociated nucleic acid; and (c) separating thedissociated nucleic acid from the solid phase, whereby the relativelyshort nucleic acid is enriched in the dissociated nucleic acid relativeto the cell-free nucleic acid from a substantially cell-free biologicalcomposition.
 3. The method of claim 2, wherein the substantiallycell-free biological composition is from a pregnant female.
 4. Themethod of claim 3, wherein the substantially cell-free biologicalcomposition is blood serum.
 5. The method of claim 3, wherein thesubstantially cell-free biological composition is blood plasma.
 6. Themethod of claim 3, wherein the substantially cell-free biologicalcomposition is urine.
 7. The method of claim 2, wherein the solid phaseis a collection of particles.
 8. The method of claim 7, wherein theparticles comprise silica.
 9. The method of claim 2, wherein the solidphase does not comprise a functional group that interacts with thenucleic acid.
 10. The method of claim 2, wherein the solid phase has anet charge.
 11. The method of claim 2, wherein the dissociationconditions comprise a volume exclusion agent.
 12. The method of claim11, wherein the volume exclusion agent comprises a polyalkyl alcohol,dextran, Ficoll, polyvinyl pyrollidone or combinations thereof.
 13. Themethod of claim 2, wherein the salt comprises an ionic salt.
 14. Themethod of claim 13, wherein the ionic salt is sodium chloride.
 15. Themethod of claim 2, wherein the dissociated nucleic acid comprisesdeoxyribonucleic acid (DNA).
 16. The method of claim 2, wherein therelatively short nucleic acid is about 200 base pairs or less and therelatively long nucleic acid is larger than about 200 base pairs. 17.The method of claim 16, wherein the relatively short nucleic acid isabout 50 to about 180 base pairs.
 18. The method of claim 2, whichfurther comprises washing the solid phase after (a).
 19. The method ofclaim 2, wherein the solid phase is paramagnetic and the dissociatednucleic acid is separated from the solid phase by a magnet.
 20. Themethod of claim 2, wherein the relatively short nucleic acid is enrichedabout 10% to about 45% in the dissociated nucleic acid relative to thecell-free nucleic acid from a substantially cell-free biologicalcomposition.
 21. The method of claim 2, wherein the solid phase isseparated from the dissociated nucleic acid by transferring thedissociated nucleic acid to an environment that does not contain thesolid phase used in (a).
 22. The method of claim 21, wherein theenvironment is a vessel.
 23. The method of claim 3, which furthercomprises detecting the presence or absence of fetal nucleic acid. 24.The method of claim 23, which further comprises detecting the presenceor absence of a chromosome abnormality.
 25. The method of claim 24,wherein the chromosome abnormality is a trisomy and the trisomy istrisomy 21, trisomy 18, trisomy 13 or combination thereof.